技术领域technical field
本发明涉及生物技术和医学领域，具体地说，本发明涉及赖氨酸特异性去甲基化酶6a(Lysine-specific deMethylase 6a，Kdm6a)在抵抗外源病毒感染，和治疗病毒感染导致的相关疾病中的效应、作用机制、实施方法和用途。The present invention relates to the fields of biotechnology and medicine, in particular, the present invention relates to the use of lysine-specific demethylase 6a (Lysine-specific deMethylase 6a, Kdm6a) in resisting exogenous virus infection and treating related diseases caused by virus infection. Effects, mechanisms of action, methods of implementation and uses in disease.
背景技术Background technique
病毒是由一种以核酸(核糖核酸RNA或脱氧核糖核酸DNA)为核心，以蛋白质为外壳(衣壳capsid)而组成的微小颗粒，其核酸可分为单股或双股，线形或球形。病毒是最小的生物病原体，从20nm到300nm不等：其中最大的是痘类病毒，例如痘苗病毒为300nm×200nm，在普通光学显微镜下勉强可见；中等大小的如流行性感冒病毒，直径为80～100nm；小型病毒如流行性乙型脑炎病毒，直径为20nm，必须在电子显微镜下才能见到(基础病毒学，莽克强等著，化学工业出版社，2005年第一版；分子病毒学，黄文林等著，人民卫生出版社，2001年第一版)。A virus is a tiny particle composed of nucleic acid (ribonucleic acid RNA or deoxyribonucleic acid DNA) as the core and protein as the shell (capsid). The nucleic acid can be divided into single strands or double strands, linear or spherical. Viruses are the smallest biological pathogens, ranging from 20nm to 300nm: the largest of which are poxviruses, such as vaccinia virus, which is 300nm × 200nm, barely visible under an ordinary light microscope; medium-sized ones such as influenza virus, with a diameter of 80 ~100nm; small viruses such as Japanese encephalitis virus, with a diameter of 20nm, must be seen under an electron microscope (Basic Virology, Mang Keqiang et al., Chemical Industry Press, 2005, first edition; Molecular Virus Science, Huang Wenlin et al., People's Health Publishing House, first edition in 2001).
随着病毒学和免疫学研究的进展，人们对病毒性疾病(Viral diseases)的认识也逐渐发展。最常用的病毒分类方式是根据核酸的性质(RNA或DNA)来对病毒进行分类，一般将病毒分为DNA病毒和RNA病毒。常见的致病性DNA病毒主要有人乳头状瘤病毒(HPV)、乙肝病毒(HBV)、EB病毒等，常见的致病性RNA病毒包括流行性出血热病毒、人获得性免疫缺陷病毒(HIV)、严重急性呼吸综合征(SARS)病毒以及流感病毒等(基础病毒学，莽克强等著，化学工业出版社，2005年第一版；分子病毒学，黄文林等著，人民卫生出版社，2001年第一版)。病毒性疾病根据临床表现及传染方式，可以分为呼吸道病毒、虫媒病毒、出疹性病毒、肠道病毒所致感染等。With the progress of virology and immunology research, people's understanding of viral diseases is gradually developing. The most commonly used virus classification method is to classify viruses according to the nature of nucleic acid (RNA or DNA). Generally, viruses are divided into DNA viruses and RNA viruses. Common pathogenic DNA viruses are mainly human papillomavirus (HPV), hepatitis B virus (HBV), Epstein-Barr virus, etc. Common pathogenic RNA viruses include epidemic hemorrhagic fever virus, human acquired immunodeficiency virus (HIV) , Severe Acute Respiratory Syndrome (SARS) Virus and Influenza Virus etc. first edition of the year). Viral diseases can be divided into infections caused by respiratory viruses, arboviruses, rash viruses, and enteroviruses according to clinical manifestations and modes of infection.
病毒性疾病往往引起比较严重的临床表现，每每引起暴发流行。病毒感染后除了短期内引起感染所引起的局部炎症外，长期的病毒感染可以导致肿瘤以及器官功能衰竭等严重的疾病，如HPV感染引起的宫颈癌、HBV感染引起的肝癌、肝硬化和肝功能衰竭、EB病毒引起的鼻咽癌、HIV引起的免疫功能缺陷和卡波济氏肉瘤、SARS引起的呼吸功能衰竭以及流感病毒引起的呼吸功能障碍等(基础病毒学，莽克强等著，化学工业出版社，2005年第一版；分子病毒学，黄文林等著，人民卫生出版社，2001年第一版)。Viral diseases often cause more serious clinical manifestations, often causing outbreaks. In addition to local inflammation caused by infection in a short period of time after viral infection, long-term viral infection can lead to serious diseases such as tumors and organ failure, such as cervical cancer caused by HPV infection, liver cancer caused by HBV infection, liver cirrhosis and liver dysfunction Failure, nasopharyngeal carcinoma caused by Epstein-Barr virus, immunodeficiency caused by HIV and Kaposi's sarcoma, respiratory failure caused by SARS, and respiratory dysfunction caused by influenza virus, etc. (basic virology, Mang Keqiang et al., Chemical Industry Press, first edition in 2005; Molecular Virology, Huang Wenlin et al., People's Health Publishing House, first edition in 2001).
每一种流行性病毒感染都可以造成极大的人体健康和国民经济的损失，然而至今对抗病毒尚无特效药，临床治疗重点就在对症和保守治疗，因此病毒性疾病的发病的分子和免疫学机理的研究和治疗策略的研究具有重大的科学意义和显示意义。Every kind of epidemic virus infection can cause great loss of human health and national economy. However, there is no specific drug against the virus so far. The focus of clinical treatment is on symptomatic and conservative treatment. Therefore, the molecular and immune mechanisms of viral diseases The study of medical mechanism and treatment strategy has great scientific significance and display significance.
干扰素(interferon，IFN)是一组体内存在的、有广泛生物活性的微量调节蛋白，具有广谱抗病毒、抗细胞增殖和免疫调节活性，已被广泛用于临床。干扰素是病毒感染后宿主细胞释放的具有抗病毒特性的糖蛋白。根据其生物学、生物化学、基因特征及细胞来源，可分为四种类型，即α、β、γ、ω型干扰素，分别来源于白细胞(单核/巨噬细胞)、成纤维细胞、淋巴细胞(T-cell和滋养层细胞)。干扰素具有抗病毒、抗增殖和免疫调节3种功能。其中α、β型IFN又称为I型干扰素，在病毒性疾病的治疗中具有重要地位。Interferon (interferon, IFN) is a group of micro-regulated proteins that exist in the body and have a wide range of biological activities. They have broad-spectrum anti-virus, anti-cell proliferation and immunomodulatory activities, and have been widely used in clinic. Interferon is a glycoprotein with antiviral properties released by host cells after virus infection. According to its biological, biochemical, genetic characteristics and cell sources, it can be divided into four types, namely α, β, γ, and ω-type interferons, which are derived from leukocytes (monocytes/macrophages), fibroblasts, Lymphocytes (T-cells and trophoblasts). Interferon has three functions of anti-virus, anti-proliferation and immune regulation. Among them, α and β type IFN are also called type I interferon, which play an important role in the treatment of viral diseases.
干扰素已经广泛应用于病毒性疾病的治疗，如带状疱疹、疱疹性口咽、小儿病毒性肺炎、流行性乙型脑炎、病毒性肝炎、艾滋病、尖锐湿疣、SARS等，可以抑制病毒的复制并调节病毒特异性免疫反应的产生，是目前已经得以证明并商业化应用于临床病毒性治疗的有效生物制剂。Interferon has been widely used in the treatment of viral diseases, such as herpes zoster, herpetic oropharynx, viral pneumonia in children, epidemic Japanese encephalitis, viral hepatitis, AIDS, genital warts, SARS, etc., can inhibit the development of viruses Replicating and regulating the generation of virus-specific immune responses is an effective biological agent that has been proven and commercialized in clinical viral therapy.
人们对于机体识别外来病毒的分子机制并不完全了解，但是随着Toll样分子的发现，机体针对病毒所产生的IFN的产生机制和作用机制逐步得到了认识。研究发现TLR3主要识别病毒分子中双链RNA(double-stranded RNA，dsRNA)(Takeuchi,O.等,Immunity.1999；11:443-451；Ozinsky,A.等,Proc Natl Acad Sci USA.2000；97:13766-13771；Alexopoulou,L.等,Nature.2001；413:732-738)；人类TLR7和TLR8基因定位于染色体Xp22，识别病毒分子中的单链RNA(single-stranded RNA，ssRNAs)(Heil,F.等,Science.2004；303:1526-1529)；人类TLR9定位于染色体3p21.3，识别细菌和病毒DNA中的CpG基序或人工合成的CpG寡核昔酸(CpG oligodeoxynucleotides，CpG ODN)(Hemmi,H.等,Nature.2000；408:740-745)。另外，病毒还可以通过TLR非依赖方式产生I型干扰素(Baccala,R.等,Nat.Med.13,543-551(2007))。其中，TLR3可以通过TRIF-TBK1/IKKε-IRF3信号传导通路诱导I型干扰素的产生；TLR7/8/9可以通过MyD88-IRF7信号通路活化诱导I型干扰素的产生；另外，一些进入细胞内的病毒可以通过RIG-I/MDA-5介导的TBK1/IKKε-IRF3信号通路活化通过TLR非依赖方式诱导I型干扰素的产生(Baccala,R.等,Nat.Med.13,543-551(2007))。能否诱导I型干扰素的产生以及能否抑制病毒的复制和扩增是检验药物治疗病毒性疾病的非常有效的标准。People do not fully understand the molecular mechanism of the body's recognition of foreign viruses, but with the discovery of Toll-like molecules, the production mechanism and action mechanism of IFN produced by the body against viruses have been gradually understood. Studies have found that TLR3 mainly recognizes double-stranded RNA (double-stranded RNA, dsRNA) in virus molecules (Takeuchi, O. et al., Immunity.1999; 11:443-451; Ozinsky, A. et al., Proc Natl Acad Sci USA.2000; 97:13766-13771; Alexopoulou, L. et al., Nature.2001; 413:732-738); human TLR7 and TLR8 genes are located on chromosome Xp22, and recognize single-stranded RNA (single-stranded RNA, ssRNAs) in virus molecules ( Heil, F. et al., Science.2004; 303:1526-1529); human TLR9 is located on chromosome 3p21.3, and recognizes CpG motifs in bacterial and viral DNA or artificially synthesized CpG oligonucleotides (CpG oligodeoxynucleotides, CpG ODN) (Hemmi, H. et al., Nature. 2000; 408:740-745). In addition, viruses can also produce type I interferon in a TLR-independent manner (Baccala, R. et al., Nat. Med. 13, 543-551 (2007)). Among them, TLR3 can induce the production of type I interferon through the TRIF-TBK1/IKKε-IRF3 signaling pathway; TLR7/8/9 can induce the production of type I interferon through the activation of MyD88-IRF7 signaling pathway; in addition, some enter the cell The virus can induce the production of type I interferon in a TLR-independent manner through RIG-I/MDA-5-mediated activation of the TBK1/IKKε-IRF3 signaling pathway (Baccala, R. et al., Nat.Med.13, 543-551 (2007 )). Whether it can induce the production of type I interferon and whether it can inhibit the replication and amplification of viruses is a very effective standard for testing drugs for treating viral diseases.
Kdm6a又名UTX，可以特异性去除H3K27me2/3的甲基基团，发挥H3K27去甲基化酶活性(Lee,MG.等,Science 318,447-450(2007))。该分子拥有高度保守的同源的C末端结构域即JMJC结构域，也是其酶活性结构域，主要发挥组蛋白去甲基化活性。该分子的N末端包含了6个重复序列的TPR结构域，主要发挥蛋白蛋白相互作用的功能。Kdm6a基因定位于X染色体，但是却不受X染色体失活的影响，在细胞和机体内广泛表达(Agger,K.等,Nature 449,731-734(2007))。Kdm6a, also known as UTX, can specifically remove the methyl group of H3K27me2/3 and exert H3K27 demethylase activity (Lee, MG. et al., Science 318, 447-450 (2007)). The molecule has a highly conserved homologous C-terminal domain, namely the JMJC domain, which is also its enzymatic activity domain, which mainly exerts histone demethylation activity. The N-terminus of the molecule contains a TPR domain with 6 repeat sequences, which mainly plays the role of protein-protein interaction. The Kdm6a gene is located on the X chromosome, but is not affected by X chromosome inactivation, and is widely expressed in cells and organisms (Agger, K. et al., Nature 449, 731-734 (2007)).
Kdm6a作为重要的表观修饰酶可以特异性去除H3K27me2/3的甲基化，在个体的分化、发育以及肿瘤组织的发生、发展过程中发挥着重要作用(Lan,F.等，Nature 449,689-694(2007))，然而在免疫系统中的功能受到的关注非常少。As an important epigenetic modification enzyme, Kdm6a can specifically remove the methylation of H3K27me2/3, and plays an important role in the differentiation and development of individuals and the occurrence and development of tumor tissues (Lan, F. et al., Nature 449, 689-694 (2007)), however its function in the immune system has received very little attention.
综上所述，本领域迫切需要开发出一种可有效抵抗病毒感染、抑制炎性因子产生的免疫学活性物质。In summary, there is an urgent need in this field to develop an immunologically active substance that can effectively resist viral infection and inhibit the production of inflammatory factors.
发明内容Contents of the invention
本发明的目的在于提供Kdm6a蛋白、Kdm6a编码序列、其促进剂或其抑制剂在调控I型干扰素产生中的用途，且进一步提供了它们在预防和治疗病毒感染性疾病中的用途。本发明的药物、药物组合物或试剂盒可用于促进I型干扰素的产生，有效抵抗外源病毒感染，和治疗病毒感染导致的相关疾病。The purpose of the present invention is to provide the use of Kdm6a protein, Kdm6a coding sequence, its promoter or its inhibitor in regulating the production of type I interferon, and further provide their use in preventing and treating viral infectious diseases. The medicine, pharmaceutical composition or kit of the present invention can be used to promote the production of type I interferon, effectively resist exogenous virus infection, and treat related diseases caused by virus infection.
本发明的第一方面，提供Kdm6a蛋白或Kdm6a编码序列、其促进剂或其抑制剂在制备用于调控I型干扰素产生的药物或试剂盒中的用途。The first aspect of the present invention provides the use of Kdm6a protein or Kdm6a coding sequence, its promoter or its inhibitor in the preparation of medicine or kit for regulating the production of type I interferon.
优选的，所述Kdm6a蛋白选自：Preferably, the Kdm6a protein is selected from:
(a)SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列；或(a) the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; or
(b)与SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列同源，其具有抑制病毒感染相关疾病和/或征状活性的蛋白或多肽；或(b) homologous to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, which has a protein or polypeptide that inhibits viral infection-related diseases and/or symptoms; or
(c)(a)或(b)的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸、且具有预防或治疗病毒感染相关疾病和/或征状活性的由(a)或(b)衍生的蛋白质或多肽。(c) Substitution, deletion or addition of one or several amino acids in the amino acid sequence of (a) or (b), and the activity of preventing or treating diseases and/or symptoms related to viral infection from (a) or (b) Derivatized protein or polypeptide.
更优选的，所述Kdm6a蛋白选自：SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列。More preferably, the Kdm6a protein is selected from the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
优选的，所述Kdm6a编码基因选自：Preferably, the Kdm6a encoding gene is selected from:
(i)SEQ ID NO：1或SEQ ID NO：1的第378-4583位序列、SEQ ID NO：3或SEQ ID NO：3的第432-4637位序列所示的核苷酸序列；或(i) SEQ ID NO: 1 or the 378-4583 sequence of SEQ ID NO: 1, SEQ ID NO: 3 or the nucleotide sequence shown in the 432-4637 sequence of SEQ ID NO: 3; or
(ii)在严格条件下与(i)限定的核苷酸序列杂交的分子；或(ii) a molecule that hybridizes under stringent conditions to the nucleotide sequence defined in (i); or
(iii)(i)或(ii)的核苷酸序列中经过取代、缺失或添加一个或几个核苷酸、且编码具有预防或治疗病毒感染相关疾病和/或征状的分子。(iii) Substitution, deletion or addition of one or several nucleotides in the nucleotide sequence of (i) or (ii) encodes a molecule capable of preventing or treating diseases and/or symptoms associated with viral infection.
更优选的，所述Kdm6a编码基因选自：SEQ ID NO：1或SEQ ID NO：1的第378-4583位序列、SEQ ID NO：3或SEQ ID NO：3的第432-4637位序列所示的核苷酸序列。More preferably, the Kdm6a encoding gene is selected from the group consisting of: SEQ ID NO: 1 or the 378-4583 sequence of SEQ ID NO: 1, SEQ ID NO: 3 or the 432-4637 sequence of SEQ ID NO: 3 The nucleotide sequence shown.
优选的，Kdm6a或其编码序列的所述促进剂选自：Kdm6a或其编码序列的过表达载体、外源性Kdm6a、Kdm6a或其编码序列的裸DNA、Kdm6a或其编码序列的脂质体包裹DNA、Kdm6a蛋白。Preferably, the promoter of Kdm6a or its coding sequence is selected from: an overexpression vector of Kdm6a or its coding sequence, exogenous Kdm6a, naked DNA of Kdm6a or its coding sequence, liposome encapsulation of Kdm6a or its coding sequence DNA, Kdm6a protein.
优选的，Kdm6a或其编码序列的所述抑制剂选自针对Kdm6a或其编码序列的：抗体、siRNA、miRNA、反义寡核苷酸、拮抗剂、阻断剂。Preferably, the inhibitor of Kdm6a or its coding sequence is selected from: antibodies, siRNA, miRNA, antisense oligonucleotides, antagonists, blocking agents against Kdm6a or its coding sequence.
优选的，所述Kdm6a蛋白是：天然纯化的蛋白、化学合成的产物、或使用重组技术从原核或真核宿主中产生，优选为人或小鼠Kdm6a，更优选为人Kdm6a蛋白。Preferably, the Kdm6a protein is: a naturally purified protein, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host using recombinant technology, preferably human or mouse Kdm6a, more preferably human Kdm6a protein.
优选的，所述宿主选自：细菌、酵母、高等动物、昆虫和哺乳动物细胞。Preferably, the host is selected from the group consisting of bacteria, yeast, higher animals, insects and mammalian cells.
优选的，所述Kdm6a、其编码序列或其促进剂促进I型干扰素的产生；所述抑制剂抑制I型干扰素的产生。所述I型干扰素选自：IFN-α或IFN-β，优选为IFN-β。Preferably, the Kdm6a, its coding sequence or its promoter promotes the production of type I interferon; the inhibitor inhibits the production of type I interferon. The type I interferon is selected from: IFN-α or IFN-β, preferably IFN-β.
优选的，所述药物或试剂盒包含Kdm6a或Kdm6a编码序列、或其促进剂，并通过促进I型干扰素的产生进一步用于预防或治疗与病毒感染相关的疾病和/或其征状；或者Preferably, the medicament or kit comprises Kdm6a or Kdm6a coding sequence, or its promoter, and is further used to prevent or treat diseases associated with viral infection and/or its symptoms by promoting the production of type I interferon; or
所述药物或试剂盒包含Kdm6a或其编码序列的抑制剂，并通过抑制I型干扰素的过度产生用于预防或治疗因I型干扰素过量表达引起的组织和器官损伤和/或其征状。The medicament or kit comprises an inhibitor of Kdm6a or its coding sequence, and is used for preventing or treating tissue and organ damage and/or symptoms thereof caused by overexpression of type I interferon by inhibiting the excessive production of type I interferon .
优选的，所述病毒感染相关疾病和/或征状为选自下组的一种或两种以上因病毒感染引起的疾病和/或征状：病毒感染后的复制；病毒感染局部的细胞(皮肤的上皮细胞、外周血的免疫细胞、免疫器官中的免疫细胞以及实体器官的组成细胞)和组织(包括皮肤、肝脏、肾脏、脑、肺脏)损伤；病毒感染造成的器官(包括皮肤、肝脏、肾脏、脑、肺脏)功能损伤。Preferably, the virus infection-related diseases and/or symptoms are one or more diseases and/or symptoms caused by virus infection selected from the following group: replication after virus infection; virus infection of local cells ( Epithelial cells of the skin, immune cells of peripheral blood, immune cells in immune organs, and constituent cells of solid organs) and tissues (including skin, liver, kidney, brain, lung) damage; organ (including skin, liver) damage caused by viral infection , kidney, brain, lung) functional impairment.
优选的，所述病毒感染相关疾病和/或征状选自：带状疱疹、疱疹性口咽、小儿病毒性肺炎、流行性乙型脑炎、病毒性肝炎、艾滋病、尖锐湿疣、SARS。Preferably, the virus infection-related diseases and/or symptoms are selected from: herpes zoster, herpetic oropharynx, pediatric viral pneumonia, Japanese encephalitis, viral hepatitis, AIDS, condyloma acuminata, and SARS.
优选的，所述病毒感染是由选自下组的一种或两种以上病毒引起的：人乳头状瘤病毒、乙肝病毒、EB病毒、流行性出血热病毒、人获得性免疫缺陷病毒、严重急性呼吸综合征病毒、流感病毒。Preferably, the viral infection is caused by one or more viruses selected from the group consisting of human papillomavirus, hepatitis B virus, Epstein-Barr virus, epidemic hemorrhagic fever virus, human acquired immunodeficiency virus, severe Acute respiratory syndrome virus, influenza virus.
优选的，所述Kdm6a蛋白及其编码序列诱导I型干扰素的产生，从而预防或治疗病毒感染相关疾病和/或征状。Preferably, the Kdm6a protein and its coding sequence induce the production of type I interferon, thereby preventing or treating diseases and/or symptoms related to viral infection.
优选的，所述药物的给药方法选自：给予Kdm6a编码序列，例如直接裸DNA注射法、脂质体包裹DNA直接注射法、金包被DNA基因枪轰击法、繁殖缺陷细菌携带质粒DNA法、复制缺陷腺病毒携带目的DNA法；给予Kdm6a蛋白，例如注射给药(如直接注射Kdm6a蛋白或用脂质体包埋的Kdm6a蛋白)、鼻腔给药、肺部给药、口服给药、透皮给药(如离子导入)。Preferably, the method of administering the drug is selected from: administering the Kdm6a coding sequence, such as direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-coated DNA gene gun bombardment, and reproduction-deficient bacteria carrying plasmid DNA. , Replication defective adenovirus carrying target DNA method; Give Kdm6a protein, such as injection (such as direct injection of Kdm6a protein or liposome-embedded Kdm6a protein), nasal administration, pulmonary administration, oral administration, permeation Transdermal administration (such as iontophoresis).
本发明的第二方面，提供了一种用于调控I型干扰素产生的药物组合物，其包含：A second aspect of the present invention provides a pharmaceutical composition for regulating the production of type I interferon, comprising:
(A)治疗有效量的Kdm6a蛋白或Kdm6a编码序列、其促进剂或其抑制剂；(A) Kdm6a protein or Kdm6a coding sequence of therapeutically effective amount, its promoter or its inhibitor;
(B)预防或治疗病毒感染相关疾病和/或征状活性的其它活性物质；以及(B) other active substances active in the prophylaxis or treatment of diseases and/or symptoms associated with viral infections; and
(C)药学上或免疫学上可接受的载体或赋形剂。(C) A pharmaceutically or immunologically acceptable carrier or excipient.
优选的，所述药物组合物中Kdm6a蛋白、其编码序列、其促进剂或其抑制剂占药物组合物总重量的0.001～99.9wt％。Preferably, the Kdm6a protein, its coding sequence, its promoter or its inhibitor in the pharmaceutical composition accounts for 0.001-99.9 wt% of the total weight of the pharmaceutical composition.
优选的，所述药物组合物中Kdm6a蛋白、其编码序列、其促进剂或其抑制剂占药物组合物总重量的1～95wt％，优选为5～90wt％，更优选10～80wt％。Preferably, the Kdm6a protein, its coding sequence, its accelerator or its inhibitor in the pharmaceutical composition accounts for 1-95wt%, preferably 5-90wt%, more preferably 10-80wt% of the total weight of the pharmaceutical composition.
优选的，在给予本发明的药物组合物之前、同时或之后，给予具有预防或治疗病毒感染相关疾病和/或征状活性的其它活性物质。所述具有预防或治疗病毒感染相关疾病和/或征状活性的其它活性物质选自：临床常用抗病毒药物(包括抗甲型流感病毒表面M2受体、抗神经氨酸苷酶、抗单磷酸次黄嘌呤核苷酸脱氢酶、抗病毒DNA多聚酶、抗HIV逆转录酶、抗HIV蛋白酶、抗唾液酸酶、抗解旋酶)的一种或多种。Preferably, other active substances having the activity of preventing or treating diseases and/or symptoms associated with viral infection are administered before, simultaneously or after administration of the pharmaceutical composition of the present invention. The other active substances that have the activity of preventing or treating viral infection-related diseases and/or symptoms are selected from: antiviral drugs commonly used in clinical practice (including anti-influenza virus surface M2 receptors, anti-neuraminidase, anti-monophosphate One or more of inosine nucleotide dehydrogenase, antiviral DNA polymerase, anti-HIV reverse transcriptase, anti-HIV protease, anti-sialidase, anti-helicase).
本发明的第三方面，提供了一种预防或治疗病毒感染相关疾病和/或其症状的方法，所述方法包括：给予需要预防或治疗的对象有效量的Kdm6a蛋白、其编码序列、和/或其促进剂、或有效量的上述的药物组合物。A third aspect of the present invention provides a method for preventing or treating viral infection-related diseases and/or symptoms thereof, the method comprising: administering an effective amount of Kdm6a protein, its coding sequence, and/or to a subject in need of prevention or treatment or an accelerator thereof, or an effective amount of the above-mentioned pharmaceutical composition.
优选的，所述病毒感染相关疾病和/或征状为选自下组的一种或多种因病毒感染引起的疾病和/或征状：病毒感染后的复制；病毒感染局部的细胞(皮肤的上皮细胞、外周血的免疫细胞、免疫器官中的免疫细胞以及实体器官的组成细胞)和组织(包括皮肤、肝脏、肾脏、脑、肺脏)损伤；病毒感染造成的器官(包括皮肤、肝脏、肾脏、脑、肺脏)功能损伤。Preferably, the virus infection-related diseases and/or symptoms are one or more diseases and/or symptoms caused by virus infection selected from the following group: replication after virus infection; virus infection of local cells (skin epithelial cells in peripheral blood, immune cells in immune organs, and constituent cells of solid organs) and tissues (including skin, liver, kidney, brain, lung) damage; organ damage caused by viral infection (including skin, liver, Kidney, brain, lung) functional impairment.
优选的，所述病毒感染是由选自下组的一种或多种病毒引起的：人乳头状瘤病毒、乙肝病毒、EB病毒、流行性出血热病毒、人获得性免疫缺陷病毒、严重急性呼吸综合征病毒、流感病毒。Preferably, the viral infection is caused by one or more viruses selected from the group consisting of human papillomavirus, hepatitis B virus, Epstein-Barr virus, epidemic hemorrhagic fever virus, human acquired immunodeficiency virus, severe acute Respiratory syndrome virus, influenza virus.
优选的，所述I型干扰素选自：IFN-α或IFN-β。Preferably, the type I interferon is selected from: IFN-α or IFN-β.
本发明的第四方面，提供了一种预防或治疗因I型干扰素过量表达引起的组织和器官损伤和/或其征状的方法，所述方法包括：给予需要预防或治疗的对象有效量的Kdm6a蛋白或其编码序列的抑制剂，或有效量的上述的药物组合物。A fourth aspect of the present invention provides a method for preventing or treating tissue and organ damage and/or its symptoms caused by overexpression of type I interferon, the method comprising: administering an effective amount to a subject in need of prevention or treatment Kdm6a protein or an inhibitor of its coding sequence, or an effective amount of the above-mentioned pharmaceutical composition.
本发明优点在于：The present invention has the advantage that:
1、本发明揭示了Kdm6a及其编码序列的新功能，即有效促进I型IFN的产生，抵抗外源病毒感染，和治疗病毒感染导致的相关疾病的功能；1. The present invention reveals the new functions of Kdm6a and its coding sequence, that is, the functions of effectively promoting the production of type I IFN, resisting exogenous virus infection, and treating related diseases caused by virus infection;
2、本发明提供了一种可有效促进I型IFN的产生，抵抗外源病毒感染，和治疗病毒感染导致的相关疾病的药物。2. The present invention provides a drug that can effectively promote the production of type I IFN, resist exogenous virus infection, and treat related diseases caused by virus infection.
附图说明Description of drawings
图1：Kdm6a真核表达载体转染小鼠腹腔巨噬细胞对VSV诱导的IFN-β产生的促进效果。其中，图1A为定量RT-PCR分析结果；图1B为ELISA分析结果。Figure 1: The promotion effect of Kdm6a eukaryotic expression vector transfected mouse peritoneal macrophages on VSV-induced IFN-β production. Among them, Fig. 1A is the result of quantitative RT-PCR analysis; Fig. 1B is the result of ELISA analysis.
图2：干扰Kdm6a在小鼠腹腔巨噬细胞的表达对VSV诱导的IFN-β产生的抑制效果。其中，图2A为定量RT-PCR分析；图2B为ELISA分析。“siCtrl”代表乱序对照干扰RNA(scrambled control RNA interference)；“siKdm6a”代表Kdm6a特异性的干扰RNA。Figure 2: The inhibitory effect of interfering with the expression of Kdm6a in mouse peritoneal macrophages on the production of IFN-β induced by VSV. Among them, Figure 2A is quantitative RT-PCR analysis; Figure 2B is ELISA analysis. "siCtrl" stands for scrambled control RNA interference; "siKdm6a" stands for Kdm6a-specific interference RNA.
具体实施方式detailed description
下面结合实施例对本发明提供的具体实施方式作详细说明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。The specific implementation modes provided by the present invention will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and changes to the present invention, and these modifications and changes are all within the scope of the present invention.
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考Sambrook等人《分子克隆实验指南》(New York：Cold Spring Harbor LaboratoryPress，1989)，或按照制造厂商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。For experimental methods not indicating specific conditions in the following examples, conventional methods in the art can be used, for example, with reference to Sambrook et al. "Molecular Cloning Experiment Guide" (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. The DNA sequencing method is a routine method in the art, and commercial companies can also provide tests.
除非另外说明，否则百分比和份数按重量计算。Percentages and parts are by weight unless otherwise indicated.
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中。文中所述的较佳实施方法与材料仅作示范之用。Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.
本发明人通过大量的研究，发现Kdm6a在病毒感染性疾病中，具有促进I型干扰素的产生的功能。在此基础上本发明人完成了本发明。Through extensive research, the inventors found that Kdm6a has the function of promoting the production of type I interferon in viral infectious diseases. The present inventors have completed the present invention on this basis.
具体而言，针对IFN调节相关基因进行应用研究是病毒分子生物学和细胞生物学研究的热点，将IFN促进基因的核苷酸和蛋白质应用于病毒性疾病的预防和治疗是人工干预病毒性感染的有效技术，因此无论是在功能基因组研究，还是病毒相关地基因治疗方面均具有广阔地应用前景。Specifically, applied research on genes related to IFN regulation is a hot spot in virus molecular biology and cell biology research. Applying nucleotides and proteins of IFN-promoting genes to the prevention and treatment of viral diseases is an important step in artificially intervening in viral infections. Therefore, it has broad application prospects in both functional genome research and virus-related gene therapy.
发明人通过研究发现过表达Kdm6a能够促进I型干扰素的产生并且该功能与其TPR结构域的相互作用功能密切相关。干扰Kdm6a的表达可以抑制I型干扰素的产生。提示Kdm6a可能具备病毒性疾病的应用前景。因此本发明还公开了应用该抗病毒分子于病毒感染性疾病的预防和治疗的方法和策略。The inventors found through research that overexpression of Kdm6a can promote the production of type I interferon and this function is closely related to the interaction function of its TPR domain. Interfering with the expression of Kdm6a can inhibit the production of type I interferons. It is suggested that Kdm6a may have application prospects in viral diseases. Therefore, the present invention also discloses methods and strategies for applying the antiviral molecule to the prevention and treatment of viral infectious diseases.
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本发明提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。All numerical ranges provided herein are intended to expressly include all values falling between the endpoints of the range and numerical ranges therebetween. Features mentioned in the invention or features mentioned in the embodiments may be combined. All the features disclosed in this specification can be used in combination with any combination, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。As used herein, "comprising", "having" or "comprising" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of Consists of ", "essentially composed of" and "consisting of" belong to the sub-concepts of "contain", "have" or "include".
Kdm6a蛋白(多肽)Kdm6a protein (polypeptide)
如本文所用，术语“Kdm6a蛋白(多肽)”、“Kdm6a蛋白(多肽)”、“Kdm6a”、“UTX”可互换使用，其一种组蛋白H3K27me2/3去甲基化酶。本发明的Kdm6a蛋白可为由SEQ ID NO：1的序列(人全长序列)或SEQ ID NO:1的第378-4583位序列(人CDS序列)或SEQ ID NO:3(小鼠全长序列)或SEQ ID NO:3的第432-4637位序列(小鼠CDS序列)所编码的蛋白质或这些蛋白质具有抗病毒作用的同源序列(例如可通过本领域已知的数据库或比对软件获得Kdm6a的同源序列)、变异体或修饰形式。例如，所述Kdm6a蛋白可选自：(a)SEQ ID NO：2或SEQ IDNO:4所示的氨基酸序列；或(b)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有抑制炎症因子的活性的由(a)衍生的蛋白质或多肽。As used herein, the terms "Kdm6a protein (polypeptide)", "Kdm6a protein (polypeptide)", "Kdm6a", "UTX", which is a histone H3K27me2/3 demethylase, are used interchangeably. The Kdm6a protein of the present invention can be composed of the sequence of SEQ ID NO: 1 (human full-length sequence) or the 378-4583 sequence of SEQ ID NO: 1 (human CDS sequence) or SEQ ID NO: 3 (mouse full-length sequence) or the protein encoded by the 432-4637th sequence (mouse CDS sequence) of SEQ ID NO: 3 or these proteins have antiviral homologous sequences (such as through databases or comparison software known in the art Homologous sequences), variants or modified forms of Kdm6a are obtained. For example, the Kdm6a protein can be selected from: (a) the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) in the amino acid sequence defined in (a) through substitution, deletion or addition of one or A protein or polypeptide derived from (a) having several amino acids and having the activity of inhibiting inflammatory factors.
本发明的蛋白质或多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等动物、昆虫和哺乳动物细胞)中产生。本发明中Kdm6a蛋白或多肽优选由人Kdm6a基因或其同源基因或家族基因编码。The proteins or polypeptides of the present invention may be purified from nature, chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher animal, insect, and mammalian cells). In the present invention, the Kdm6a protein or polypeptide is preferably encoded by human Kdm6a gene or its homologous gene or family gene.
本发明蛋白质或多肽的变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个，例如1、2、3、4、5、6、7、8、9或10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质或多肽的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质或多肽的功能，例如本发明的Kdm6a蛋白质或多肽可包括或不包括起始的甲硫氨酸残基而仍然具有抗病毒活性。Variations of the protein or polypeptide of the present invention include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10 deletion, insertion and/or substitution of amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide. For another example, adding one or several amino acids at the C-terminal and/or N-terminal will generally not change the function of the protein or polypeptide, for example, the Kdm6a protein or polypeptide of the present invention may or may not include the initial methionine residue and still have antiviral activity.
可采用辐射或暴露于诱变剂下来产生随机诱变，也可通过定点诱变法或其它已知的分子生物学技术来获得上述(b)中的蛋白质或多肽。可利用编码所述蛋白质或多肽的编码序列来构建转基因动物，并观察该转基因动物对病毒感染的抗性是否有所改良来筛选和鉴别所得蛋白质或多肽。The protein or polypeptide in (b) above can be obtained by radiation or exposure to mutagen to produce random mutagenesis, or by site-directed mutagenesis or other known molecular biology techniques. The coding sequence encoding the protein or polypeptide can be used to construct a transgenic animal, and observe whether the resistance of the transgenic animal to virus infection is improved to screen and identify the resulting protein or polypeptide.
根据重组生产方案所用的宿主，本发明的蛋白质或多肽可以是糖基化的，或可以是非糖基化的。该术语还包括Kdm6a蛋白的活性片段和活性衍生物。Depending on the host used in the recombinant production protocol, the protein or polypeptide of the invention may be glycosylated, or may be non-glycosylated. The term also includes active fragments and active derivatives of the Kdm6a protein.
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与Kdm6a蛋白编码序列杂交的序列所编码的蛋白、以及利用抗Kdm6a蛋白的抗血清获得的多肽或蛋白。本发明还可使用其它多肽，如包含Kdm6a蛋白或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了Kdm6a蛋白的可溶性片段。通常，该片段具有Kdm6a蛋白序列的至少约10个连续氨基酸，通常至少约30个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, encoded by sequences that can hybridize with the Kdm6a protein coding sequence under high or low stringency conditions Protein, and the polypeptide or protein obtained by using anti-Kdm6a protein antiserum. The present invention can also use other polypeptides, such as fusion proteins comprising Kdm6a protein or fragments thereof. In addition to the nearly full-length polypeptides, the present invention also includes soluble fragments of the Kdm6a protein. Usually, the fragment has at least about 10 contiguous amino acids of the Kdm6a protein sequence, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids. consecutive amino acids.
Kdm6a蛋白编码基因Kdm6a protein coding gene
如本文所用，术语“Kdm6a基因”或“Kdm6a蛋白编码序列”可互换使用，均是指一种编码本发明所述的Kdm6a蛋白或多肽的序列，其可为SEQ ID NO：1(人全长)或SEQ ID NO：1的第378-4583位(人CDS)序列、SEQ ID NO：3(小鼠全长)或SEQ ID NO：3的第432-4637位(小鼠CDS)序列所示的核苷酸序列、在严格条件下与这些序列杂交的分子、或与上述分子高度同源的家族基因分子，所述基因的表达对病毒感染具有一定的抑制作用。本发明的Kdm6a基因可选自：(i)SEQ ID NO：1或SEQ ID NO：1的第378-4583位序列、SEQ ID NO：3或SEQ IDNO：3的第432-4637位序列所示的核苷酸序列；或(ii)在严格条件下与(i)限定的序列杂交且具有抑制炎症因子活性的分子。As used herein, the term "Kdm6a gene" or "Kdm6a protein coding sequence" can be used interchangeably, and both refer to a sequence encoding the Kdm6a protein or polypeptide of the present invention, which can be SEQ ID NO: 1 (human whole long) or SEQ ID NO: 1's 378-4583 (human CDS) sequence, SEQ ID NO: 3 (mouse full length) or SEQ ID NO: 3's 432-4637 (mouse CDS) sequence The nucleotide sequences shown, molecules that hybridize to these sequences under stringent conditions, or family gene molecules that are highly homologous to the above molecules, the expression of the genes has a certain inhibitory effect on virus infection. The Kdm6a gene of the present invention can be selected from: (i) SEQ ID NO: 1 or the 378-4583 sequence of SEQ ID NO: 1, SEQ ID NO: 3 or shown in the 432-4637 sequence of SEQ ID NO: 3 or (ii) a molecule that hybridizes to the sequence defined in (i) under stringent conditions and has the activity of inhibiting inflammatory factors.
如本文所用，术语“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在50％，优选55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上或90％以上，更优选是95％以上时才发生杂交。例如，所述序列可为(a)中所限定序列的互补序列。As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only the identity between the two sequences is at least 50%, preferably 55% More than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, more preferably more than 95%, hybridization occurs. For example, the sequence may be the complement of the sequence defined in (a).
本发明的Kdm6a基因核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。The Kdm6a gene nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.
应理解，本发明的Kdm6a基因优选获自人，获自其它动物的与人Kdm6a基因高度同源(如具有50％以上，优选55％以上、60％以上、65％以上、70％以上、75％以上、80％以上，更优选85％以上如85％、90％、95％、甚至98％序列相同性)的其它基因也在本发明优选考虑的等同范围之内。比对序列相同性的方法和工具也是本领域周知的，如BLAST。It should be understood that the Kdm6a gene of the present invention is preferably obtained from humans, and those obtained from other animals are highly homologous to the human Kdm6a gene (such as having more than 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%) % or more, more than 80%, more preferably more than 85%, such as 85%, 90%, 95%, or even 98% sequence identity) are also within the equivalent scope of the present invention. Methods and tools for aligning sequence identities are also well known in the art, such as BLAST.
Kdm6a或其编码序列的促进剂和抑制剂Promoters and inhibitors of Kdm6a or its coding sequence
本发明中还涉及Kdm6a或其编码序列的“促进剂”。术语“促进剂”或“Kdm6a或其编码序列的促进剂”可互换使用，是指可提高Kdm6a或其编码序列的水平或活性的物质。可用于本发明中的促进剂包括但不限于：Kdm6a表达载体、外源性Kdm6a、Kdm6a或其编码序列的裸DNA、Kdm6a或其编码序列的脂质体包裹DNA、Kdm6a蛋白。"Promoters" of Kdm6a or its coding sequence are also contemplated in the present invention. The terms "promoter" or "promoter of Kdm6a or its coding sequence" are used interchangeably and refer to substances that increase the level or activity of Kdm6a or its coding sequence. Promoters that can be used in the present invention include but are not limited to: Kdm6a expression vector, exogenous Kdm6a, naked DNA of Kdm6a or its coding sequence, liposome-encapsulated DNA of Kdm6a or its coding sequence, Kdm6a protein.
本发明的Kdm6a、其编码序列或其促进剂促进I型干扰素的产生进一步用于预防或治疗与病毒感染相关的疾病和/或其征状；The Kdm6a of the present invention, its coding sequence or its promoter promotes the production of type I interferon and is further used for preventing or treating diseases related to viral infection and/or its symptoms;
本发明中还涉及Kdm6a或其编码序列的“抑制剂”。术语“抑制剂”或“Kdm6a或其编码序列的抑制剂”可互换使用，是指可降低Kdm6a或其编码序列的水平或活性的物质。可用于本发明中的抑制剂包括但不限于，针对Kdm6a或其编码序列的：抗体、siRNA、miRNA、反义寡核苷酸、拮抗剂、阻断剂。"Inhibitors" of Kdm6a or its coding sequence are also contemplated in the present invention. The terms "inhibitor" or "inhibitor of Kdm6a or its coding sequence" are used interchangeably and refer to a substance that reduces the level or activity of Kdm6a or its coding sequence. Inhibitors that can be used in the present invention include, but are not limited to, against Kdm6a or its coding sequence: antibodies, siRNA, miRNA, antisense oligonucleotides, antagonists, blocking agents.
本发明的抑制剂可抑制I型干扰素的过度产生用于预防或治疗因I型干扰素过量表达引起的组织和器官损伤和/或其征状。The inhibitor of the present invention can inhibit the excessive production of type I interferon and is used for preventing or treating tissue and organ damage and/or symptoms caused by excessive expression of type I interferon.
载体、宿主及转基因动物Vectors, hosts and transgenic animals
本发明还涉及包含Kdm6a基因的载体，以及用该载体经基因工程产生的宿主细胞，以及通过转基因获得高表达Kdm6a的转基因动物。The invention also relates to a vector containing Kdm6a gene, a host cell produced by genetic engineering using the vector, and a transgenic animal with high Kdm6a expression through transgene.
通过常规的重组DNA技术(Science，1984；224：1431)，可利用本发明的编码序列可用来表达或生产重组的Kdm6a蛋白。一般来说有以下步骤：By conventional recombinant DNA technology (Science, 1984; 224: 1431), the coding sequence of the present invention can be used to express or produce recombinant Kdm6a protein. Generally speaking, there are the following steps:
(1)用本发明的编码Kdm6a蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；(1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the Kdm6a protein of the present invention, or with a recombinant expression vector containing the polynucleotide;
(2)在合适的培养基中培养的宿主细胞；和(2) host cells cultured in a suitable medium; and
(3)从培养基或细胞中分离、纯化蛋白质或多肽。(3) Separation and purification of protein or polypeptide from culture medium or cells.
本发明中，术语“载体”与“重组表达载体”可互换使用，指本领域熟知的细菌质粒、噬菌体、酵母质粒、动物细胞病毒、哺乳动物细胞病毒或其它载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。In the present invention, the term "vector" and "recombinant expression vector" are used interchangeably, and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.
本领域的技术人员熟知的方法能用于构建含Kdm6a编码序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。本发明中优选使用pcDNA3.1载体、pIRES2-EGFP载体、Adeno-X表达系统和pSilencer 2.1-U6-neo干扰载体。Methods well known to those skilled in the art can be used to construct an expression vector containing the Kdm6a coding sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In the present invention, pcDNA3.1 vector, pIRES2-EGFP vector, Adeno-X expression system and pSilencer 2.1-U6-neo interference vector are preferably used.
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质或多肽。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如动物细胞。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；动物细胞等。在本发明中，优选采用大肠杆菌细菌细胞、小鼠巨噬细胞作为宿主细胞。Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins or polypeptides. The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells and the like. In the present invention, Escherichia coli bacterial cells and mouse macrophages are preferably used as host cells.
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.
本发明中术语“转基因动物”、或“转化动物”可互换使用，均指通过常规转基因的方法获得的转入本发明Kdm6a基因并稳定高表达Kdm6a蛋白或多肽的细胞、器官、组织或个体。In the present invention, the term "transgenic animal" or "transformed animal" can be used interchangeably, and they all refer to cells, organs, tissues or individuals that are transferred to the Kdm6a gene of the present invention and stably and highly express Kdm6a protein or polypeptide obtained by conventional transgenic methods .
在上面的方法中的重组多肽可在细胞内或在细胞膜上表达或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The recombinant polypeptide in the above method can be expressed inside the cell or on the cell membrane or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
药物、药物组合物或试剂盒Drug, pharmaceutical composition or kit
本发明还提供了一种药物、药物组合物或试剂盒，其中含有有效量的本发明的Kdm6a或Kdm6a编码序列、其促进剂或抑制剂，以及药学上或免疫学上可接受的载体。如本文所用，术语“活性物质”或“本发明的活性物质”可互换使用，是指Kdm6a或Kdm6a编码序列、其促进剂或抑制剂。The present invention also provides a medicament, a pharmaceutical composition or a kit, which contains an effective amount of the Kdm6a or Kdm6a coding sequence of the present invention, its promoter or inhibitor, and a pharmaceutically or immunologically acceptable carrier. As used herein, the terms "active substance" or "active substance of the present invention" are used interchangeably to refer to Kdm6a or a Kdm6a coding sequence, a promoter or an inhibitor thereof.
在较佳的实施方案中，所述药物组合物可用于治疗与现有技术中已知可治疗或预防病毒感染相关疾病和/或征状，例如：病毒感染后的复制；病毒感染后的复制；病毒感染局部的细胞(皮肤的上皮细胞、外周血的免疫细胞、免疫器官中的免疫细胞以及实体器官的组成细胞)和组织(包括皮肤、肝脏、肾脏、脑、肺脏)损伤；病毒感染造成的器官(包括皮肤、肝脏、肾脏、脑、肺、脾脏)功能损伤。In a preferred embodiment, the pharmaceutical composition can be used to treat diseases and/or symptoms associated with known treatable or preventable viral infections in the prior art, for example: replication after viral infection; replication after viral infection ; Virus infects local cells (skin epithelial cells, peripheral blood immune cells, immune cells in immune organs, and constituent cells of solid organs) and tissue (including skin, liver, kidney, brain, lung) damage; viral infection causes Impairment of organ function (including skin, liver, kidney, brain, lung, spleen).
在本发明中，所述病毒感染相关疾病和/或征状可选自：带状疱疹、疱疹性口咽、小儿病毒性肺炎、流行性乙型脑炎、病毒性肝炎、艾滋病、尖锐湿疣、SARS。In the present invention, the virus infection-related diseases and/or symptoms may be selected from: herpes zoster, herpetic oropharynx, viral pneumonia in children, Japanese encephalitis, viral hepatitis, AIDS, condyloma acuminata, SARS.
病毒感染可由选自下组的一种或多种病毒引起的：人乳头状瘤病毒、乙肝病毒、EB病毒、流行性出血热病毒、人获得性免疫缺陷病毒、严重急性呼吸综合征病毒、流感病毒。Viral infection may be caused by one or more viruses selected from the group consisting of human papillomavirus, hepatitis B virus, Epstein-Barr virus, epidemic hemorrhagic fever virus, human acquired immunodeficiency virus, severe acute respiratory syndrome virus, influenza Virus.
如本文所用，术语“含有”或“包括”包括了“包含”、“基本上由……构成”、和“由……构成”。As used herein, the terms "comprising" or "comprising" include "comprising", "consisting essentially of", and "consisting of".
如本文所用，术语“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。As used herein, the term "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and/or animals without undue adverse side effects such as toxicity, irritation and allergic reactions, ie having a reasonable benefit/risk ratio.
如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。As used herein, the term "effective amount" refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals.
如本文所用，术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在《雷明顿药物科学》(Remington’s Pharmaceutical Sciences，Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
在组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如填充剂、崩解剂、润滑剂、助流剂、泡腾剂、润湿剂或乳化剂、矫味剂、pH缓冲物质等。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。Pharmaceutically acceptable carriers in compositions can contain liquids such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, and the like. Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8.
本发明的组合物中Kdm6a蛋白或其编码序列有效成分占组合物总重量的0.001～99.9wt％；优选为组合物总重量的1～95wt％，较优选为5～90wt％，更优选10～80wt％。余量为药学上可接受的载体以及其它添加剂等物质。In the composition of the present invention, the active ingredient of the Kdm6a protein or its coding sequence accounts for 0.001 to 99.9 wt% of the total weight of the composition; preferably 1 to 95 wt% of the total weight of the composition, more preferably 5 to 90 wt%, more preferably 10 to 90 wt%. 80 wt%. The balance is pharmaceutically acceptable carrier and other additives and other substances.
如本文所用，术语“单位剂型”是指为了服用方便，将本发明的组合物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。As used herein, the term "unit dosage form" refers to the preparation of the composition of the present invention into a dosage form required for single administration for convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, and capsule dosage forms. , Sustained release agent.
在本发明的另一优选实施方式中，所述组合物为单位剂型或多剂型，且其中Kdm6a蛋白或其编码序列的含量为0.01～2000mg/剂，优选0.1～1500mg/剂，更优选1～1000mg/剂。在本发明的另一个优选例中，每天施用1～6剂本发明的组合物，优选施用1～3剂；最优选的，每天服用的剂量为1剂。In another preferred embodiment of the present invention, the composition is in unit dosage form or multiple dosage form, and the content of Kdm6a protein or its coding sequence is 0.01-2000 mg/dose, preferably 0.1-1500 mg/dose, more preferably 1-2000 mg/dose. 1000mg/dose. In another preferred embodiment of the present invention, 1-6 doses of the composition of the present invention are administered per day, preferably 1-3 doses are administered; most preferably, the daily dose is 1 dose.
应理解，所用Kdm6a蛋白或其编码序列的有效剂量可随待施用或治疗的对象的严重程度而变化。具体情况根据对象的个体情况(例如对象体重、年龄、身体状况、所需达到的效果)来决定，这在熟练医师可以判断的范围内。It should be understood that the effective dose of Kdm6a protein or its coding sequence used may vary with the severity of the subject to be administered or treated. The specific situation is determined according to the individual conditions of the subject (such as the subject's weight, age, physical condition, desired effect), which is within the scope of judgment of a skilled physician.
本发明的组合物，可以为固态(如颗粒剂、片剂、冻干粉、栓剂、胶囊、舌下含片)或液态(如口服液)或其它合适的形状。给药途径可采用：(1)直接裸DNA或者蛋白质注射法；(2)将Kdm6a的cDNA、mRNA和蛋白质与转铁蛋白/多聚L-赖氨酸复合物连接，以增强其生物效应；(3)cDNA、mRNA和蛋白质与带正电荷的脂类形成复合物，以克服磷酸骨架负电荷所致的穿越细胞膜的困难；(4)用脂质体包裹cDNA、mRNA和蛋白质后介导进入细胞，既有利于大分子的顺利进入又免受细胞外各种酶的水解作用；(5)cDNA、mRNA和蛋白质与胆固醇结合使其胞浆保持时间增加10倍；(6)用免疫脂质体转运cDNA、mRNA和蛋白质可使其特异性转运至靶组织和靶细胞；(7)将cDNA、mRNA和蛋白质体外转染给转载细胞(如成纤维细胞)也可较好地将Kdm6a相关药物载入靶细胞内；(8)电打孔(electroporation)，即借助于电流将cDNA、mRNA和蛋白质导入靶细胞。The composition of the present invention can be solid (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable shapes. The route of administration can be: (1) direct naked DNA or protein injection; (2) link the cDNA, mRNA and protein of Kdm6a to the transferrin/poly-L-lysine complex to enhance its biological effect; (3) cDNA, mRNA and protein form a complex with positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) mediate entry after encapsulating cDNA, mRNA and protein with liposomes Cells, which are conducive to the smooth entry of macromolecules and are free from the hydrolysis of various extracellular enzymes; (5) the combination of cDNA, mRNA and protein with cholesterol increases the retention time of the cytoplasm by 10 times; (6) the use of immune lipids (7) Transfection of cDNA, mRNA and protein to transfected cells (such as fibroblasts) in vitro can also better transfer Kdm6a-related drugs Loading into target cells; (8) electroporation, that is, introducing cDNA, mRNA and protein into target cells by means of electric current.
此外，本发明的组合物中还可含有用于改善和治疗病毒感染性疾病的其它活性物质，所述的其它活性物质选自下组：临床常用抗病毒药物(包括抗甲型流感病毒表面M2受体、抗神经氨酸苷酶、抗单磷酸次黄嘌呤核苷酸脱氢酶、抗病毒DNA多聚酶、抗HIV逆转录酶、抗HIV蛋白酶、抗唾液酸酶、抗解旋酶)的一种或多种。In addition, the composition of the present invention can also contain other active substances for improving and treating viral infectious diseases, and the other active substances are selected from the following group: commonly used clinical antiviral drugs (including anti-influenza virus surface M2 receptor, anti-neuraminidase, anti-inosine nucleotide dehydrogenase, anti-viral DNA polymerase, anti-HIV reverse transcriptase, anti-HIV protease, anti-sialidase, anti-helicase) one or more species.
本发明的Kdm6a相关的核苷酸和蛋白质药物相互间可以联合应用，还可以与其它药物和治疗手段联合，用于病毒感染性疾病的预防和治疗。The Kdm6a-related nucleotide and protein drugs of the present invention can be used in combination with each other, and can also be used in combination with other drugs and therapeutic means for the prevention and treatment of viral infectious diseases.
实施例1：Kdm6a对巨噬细胞IFN-β产生的促进作用Example 1: The promotion effect of Kdm6a on the production of macrophage IFN-β
腹腔注射3％的脱水硫羟乙酸培养基2ml，4天后，颈椎脱臼法处死小鼠，75％酒精浸泡5-10分钟，小鼠腹部朝上，在腹中线剪开一小口，撕开皮肤，完全暴露腹膜。然后用20ml注射器注入10ml无血清1640培养基，针尖于腹膜下至腹腔则边，且针尖朝上，避开肠子和脂肪，缓慢抽吸，此时，腹腔灌洗液即含有大量巨噬细胞。将收集的腹腔液放于50ml的离心管。重复此步骤一次，共20ml。细胞悬液800g离心5分钟，弃上清，用含有10％FCS的DMEM重悬。细胞悬液置入培养板，37℃培养1小时后换液，贴壁细胞即为新鲜分离的小鼠腹腔巨噬细胞。Intraperitoneal injection of 2ml of 3% dehydrated thiolacetic acid medium, 4 days later, the mice were killed by cervical dislocation, soaked in 75% alcohol for 5-10 minutes, the abdomen of the mice was facing up, a small mouth was cut in the midline of the abdomen, and the skin was torn open. Fully expose the peritoneum. Then use a 20ml syringe to inject 10ml of serum-free 1640 medium. The needle tip is placed from the subperitoneum to the side of the peritoneal cavity, and the needle tip is facing upwards, avoiding the intestines and fat, and slowly sucking. At this time, the peritoneal lavage fluid contains a large number of macrophages. Put the collected peritoneal fluid into a 50ml centrifuge tube. Repeat this step once for a total of 20ml. Centrifuge the cell suspension at 800 g for 5 minutes, discard the supernatant, and resuspend with DMEM containing 10% FCS. The cell suspension was put into the culture plate, cultured at 37°C for 1 hour, and then the medium was changed. The adherent cells were freshly isolated mouse peritoneal macrophages.
采用pcDNA3.1(购自Invitrogen公司)作为真核表达载体，将带有XhoI/BamHI酶切位点的Kdm6a的SEQ ID NO:3的cDNA产物插入该载体的相应位置，在E.Coli菌株DH-5α内扩增该载体，测序鉴定后纯化得到Kdm6a的真核表达载体。瞬时转染(转染试剂JetPei-Macrophage购自Polyplus公司)小鼠巨噬细胞。48小时后，加入0.1MOI的活VSV病毒(Indiana Strain)(可购自ATCC，也可采用下述文献方法制备：Li X,Zhang Q,Ding Y,LiuY,Zhao D,Zhao K,Shen Q,Liu X,Zhu X,Li N,Cheng Z,Fan G,Wang Q,CaoX.Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate TBK1 foractivation of antiviral innate immunity.Nature Immunology.2016；17(7):806-815.)处理不同时间后，收集细胞和培养上清，制备cDNA用于定量RT-PCR(94℃，30Sec；58℃，30Sec；72℃，30Sec；共30循环)检测IFN-β mRNA水平；或者用培养上清通过ELISA(所用试剂盒购自PBL公司)检测IFN-β蛋白水平。Using pcDNA3.1 (purchased from Invitrogen Company) as a eukaryotic expression vector, the cDNA product of SEQ ID NO: 3 of Kdm6a with XhoI/BamHI restriction site was inserted into the corresponding position of the vector, in E.Coli bacterial strain DH The vector was amplified in -5α, sequenced and identified, and purified to obtain the eukaryotic expression vector of Kdm6a. Mouse macrophages were transiently transfected (transfection reagent JetPei-Macrophage was purchased from Polyplus). After 48 hours, add 0.1 MOI of live VSV virus (Indiana Strain) (can be purchased from ATCC, and can also be prepared by the following literature methods: Li X, Zhang Q, Ding Y, LiuY, Zhao D, Zhao K, Shen Q, Liu X, Zhu X, Li N, Cheng Z, Fan G, Wang Q, Cao X. Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate TBK1 for activation of antiviral innate immunity. Nature Immunology. 2016; 17(7):806-815.) Different treatment After a period of time, collect the cells and culture supernatant, and prepare cDNA for quantitative RT-PCR (94°C, 30Sec; 58°C, 30Sec; 72°C, 30Sec; a total of 30 cycles) to detect IFN-β mRNA levels; or use the culture supernatant The level of IFN-β protein was detected by ELISA (the kit used was purchased from PBL Company).
IFN-β mRNA的定量RT-PCR分析和IFN-β的ELISA分析的结果分别如图1A和图1B所示。结果显示：Kdm6a真核表达载体能够促进小鼠巨噬细胞IFN-β的产生。The results of quantitative RT-PCR analysis of IFN-β mRNA and ELISA analysis of IFN-β are shown in Figure 1A and Figure 1B, respectively. The results showed that: Kdm6a eukaryotic expression vector can promote the production of IFN-β in mouse macrophages.
实施例2：干扰Kdm6a表达对巨噬细胞IFN-β产生的抑制作用Example 2: The inhibitory effect of interfering with Kdm6a expression on the production of IFN-β in macrophages
用针对Kdm6a的干扰RNA(Si-Kdm6a)及模拟物对照(Si-模拟物)转染(转染试剂INTERFERin购自Polyplus公司)如上所得的小鼠巨噬细胞，6小时后更换新鲜RPMI 1640培养基。The mouse macrophages obtained above were transfected with the interfering RNA (Si-Kdm6a) against Kdm6a (Si-Kdm6a) and the mock control (Si-mimetic) (the transfection reagent INTERFERin was purchased from Polyplus Company), and cultured in fresh RPMI 1640 after 6 hours base.
针对Kdm6a的干扰RNA(Si-Kdm6a)及模拟物对照(Si-模拟物)购自Genephama公司，Si-Kdm6a的序列如SEQ ID NO：5和SEQ ID NO：6所示，Si-模拟物的序列如SEQ ID NO：7和SEQ ID NO：8所示，合成时在3'增加2个dT使序列更加稳定。The interfering RNA (Si-Kdm6a) against Kdm6a and the mimic control (Si-mimetic) were purchased from Genephama Company, the sequence of Si-Kdm6a is shown in SEQ ID NO: 5 and SEQ ID NO: 6, and the Si-mimetic The sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8. During synthesis, 2 dTs were added at the 3' to make the sequences more stable.
具体序列如下：The specific sequence is as follows:
Si-Kdm6a序列：Si-Kdm6a sequence:
5'-GCUGCUACGAAUCUCUAAUTT-3'(顺义)；5'-GCUGCUACGAAUCUCUAAUTT-3' (Shunyi);
5'-AUUAGAGAUUCGUAGCAGCTT-3'(反义)。5'-AUUAGAGAUUCGUAGCAGCTT-3' (antisense).
Si-模拟物序列：Si-mimetic sequence:
5'-UUCUCCGAACGUGUCACGUTT-3'(顺义)；5'-UUCUCCGAACGUGUCACGUTT-3' (Shunyi);
5'-ACGUGACACGUUCGGAGAATT-3′(反义)。5'-ACGUGACACGUUCGGAGAATT-3' (antisense).
48小时后，加入0.1MOI的活VSV病毒(可购自ATCC，也可采用下述文献方法制备：LiX,Zhang Q,Ding Y,Liu Y,Zhao D,Zhao K,Shen Q,Liu X,Zhu X,Li N,Cheng Z,Fan G,Wang Q,Cao X.Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate TBK1for activation of antiviral innate immunity.Nature Immunology.2016；17(7):806-815.)处理不同时间后，收集细胞和培养上清，制备cDNA用于定量RT-PCR(94℃，30Sec；58℃，30Sec；72℃，30Sec；共30循环)检测Kdm6a mRNA水平,IFN-β mRNA水平；或者用培养上清通过ELISA(所用试剂盒购自PBL公司)检测IFN-β蛋白水平。After 48 hours, add the live VSV virus of 0.1MOI (can be purchased from ATCC, also can adopt following literature method to prepare: LiX, Zhang Q, Ding Y, Liu Y, Zhao D, Zhao K, Shen Q, Liu X, Zhu X, Li N, Cheng Z, Fan G, Wang Q, Cao X. Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate TBK1 for activation of antiviral innate immunity. Nature Immunology. 2016; 17 (7): 806-815.) after different treatment time, Collect cells and culture supernatant, prepare cDNA for quantitative RT-PCR (94°C, 30Sec; 58°C, 30Sec; 72°C, 30Sec; a total of 30 cycles) to detect Kdm6a mRNA level, IFN-β mRNA level; The protein level of IFN-β was detected by ELISA (the kit used was purchased from PBL Company).
Kdm6a mRNA水平，IFN-β mRNA的定量RT-PCR分析和IFN-β的ELISA分析的结果分别如图2A和图2B所示。结果显示：干扰Kdm6a在小鼠巨噬细胞的表达对IFN-β产生起抑制作用。The results of Kdm6a mRNA level, quantitative RT-PCR analysis of IFN-β mRNA and ELISA analysis of IFN-β are shown in Figure 2A and Figure 2B, respectively. The results showed that interfering with the expression of Kdm6a in mouse macrophages inhibited the production of IFN-β.
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. These equivalent modifications or replacements are all included within the scope defined by the claims of the present application.
SEQUENCE LISTINGSEQUENCE LISTING
<110> 中国人民解放军第二军医大学<110> The Second Military Medical University of the Chinese People's Liberation Army
<120> 一种新型抗病毒分子Kdm6a的抗病毒作用及其应用<120> Antiviral effect and application of a novel antiviral molecule Kdm6a
<130> /<130> /
<160> 8<160> 8
<170> PatentIn version 3.3<170> PatentIn version 3.3
<210> 1<210> 1
<211> 5785<211> 5785
<212> DNA<212>DNA
<213> 智人(Homo sapiens)<213> Homo sapiens
<220><220>
<221> CDS<221> CDS
<222> (378)..(4583)<222> (378)..(4583)
<400> 1<400> 1
gtgtgacaca attacaacaa ctttgtgctg gtgccgggga agtttgtgtc tccaacgaat 60gtgtgacaca attacaacaa ctttgtgctg gtgccgggga agtttgtgtc tccaacgaat 60
cccctcagtg ctccccagcc ccgcgcgctc cggccgttcc cgccgtcccc gcctgtggct 120cccctcagtg ctccccagcc ccgcgcgctc cggccgttcc cgccgtcccc gcctgtggct 120
gccccctgcc caaccccgcg atgtgaccct acagccgaaa gccgccgctg ccgacccggg 180gccccctgcc caaccccgcg atgtgaccct acagccgaaa gccgccgctg ccgacccggg 180
ggctccgcag cccctgccgc cgccgccgcc gccttcaccg ccgccgcgtt gggatttttc 240ggctccgcag cccctgccgc cgccgccgcc gccttcaccg ccgccgcgtt gggatttttc 240
gtcgccgccg cccgcggcgg aggaggaggc ggcgataaag ttggtgtgct ggtcccgcgc 300gtcgccgccg cccgcggcgg aggaggaggc ggcgataaag ttggtgtgct ggtcccgcgc 300
gcagattggg ggcgtcactg cgggccccgg tccgaggggg ggtgtcggcg ttggagttgt 360gcagattggg ggcgtcactg cgggccccgg tccgaggggggggtgtcggcg ttggagttgt 360
gaattcgctg cgtttcc atg aaa tcc tgc gga gtg tcg ctc gct acc gcc 410gaattcgctg cgtttcc atg aaa tcc tgc gga gtg tcg ctc gct acc gcc 410
Met Lys Ser Cys Gly Val Ser Leu Ala Thr Ala Met Lys Ser Cys Gly Val Ser Leu Ala Thr Ala
1 5 10 1 5 10
gcc gct gcc gcc gcc gct ttc ggt gat gag gaa aag aaa atg gcg gcg 458gcc gct gcc gcc gcc gct ttc ggt gat gag gaa aag aaa atg gcg gcg 458
Ala Ala Ala Ala Ala Ala Phe Gly Asp Glu Glu Lys Lys Met Ala AlaAla Ala Ala Ala Ala Ala Phe Gly Asp Glu Glu Lys Lys Met Ala Ala
15 20 25 15 20 25
gga aaa gcg agc ggc gag agc gag gag gcg tcc ccc agc ctg aca gcc 506gga aaa gcg agc ggc gag agc gag gag gcg tcc ccc agc ctg aca gcc 506
Gly Lys Ala Ser Gly Glu Ser Glu Glu Ala Ser Pro Ser Leu Thr AlaGly Lys Ala Ser Gly Glu Ser Glu Glu Ala Ser Pro Ser Leu Thr Ala
30 35 40 30 35 40
gag gag agg gag gcg ctc ggc gga ctg gac agc cgc ctc ttt ggg ttc 554gag gag agg gag gcg ctc ggc gga ctg gac agc cgc ctc ttt ggg ttc 554
Glu Glu Arg Glu Ala Leu Gly Gly Leu Asp Ser Arg Leu Phe Gly PheGlu Glu Arg Glu Ala Leu Gly Gly Leu Asp Ser Arg Leu Phe Gly Phe
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gtg aga ttt cat gaa gat ggc gcc agg acg aag gcc cta ctg ggc aag 602gtg aga ttt cat gaa gat ggc gcc agg ag aag gcc cta ctg ggc aag 602
Val Arg Phe His Glu Asp Gly Ala Arg Thr Lys Ala Leu Leu Gly LysVal Arg Phe His Glu Asp Gly Ala Arg Thr Lys Ala Leu Leu Gly Lys
60 65 70 7560 65 70 75
gct gtt cgc tgc tat gaa tct cta atc tta aaa gct gaa gga aaa gtg 650gct gtt cgc tgc tat gaa tct cta atc tta aaa gct gaa gga aaa gtg 650
Ala Val Arg Cys Tyr Glu Ser Leu Ile Leu Lys Ala Glu Gly Lys ValAla Val Arg Cys Tyr Glu Ser Leu Ile Leu Lys Ala Glu Gly Lys Val
80 85 90 80 85 90
gag tct gat ttc ttt tgt caa tta ggt cac ttc aac ctc tta ttg gaa 698gag tct gat ttc ttt tgt caa tta ggt cac ttc aac ctc tta ttg gaa 698
Glu Ser Asp Phe Phe Cys Gln Leu Gly His Phe Asn Leu Leu Leu GluGlu Ser Asp Phe Phe Cys Gln Leu Gly His Phe Asn Leu Leu Leu Glu
95 100 105 95 100 105
gat tat cca aaa gca tta tct gca tac cag agg tac tac agt tta cag 746gat tat cca aaa gca tta tct gca tac cag agg tac tac agt tta cag 746
Asp Tyr Pro Lys Ala Leu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu GlnAsp Tyr Pro Lys Ala Leu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu Gln
110 115 120 110 115 120
tct gac tac tgg aag aat gct gcc ttt tta tat ggt ctt ggt ttg gtc 794tct gac tac tgg aag aat gct gcc ttt tta tat ggt ctt ggt ttg gtc 794
Ser Asp Tyr Trp Lys Asn Ala Ala Phe Leu Tyr Gly Leu Gly Leu ValSer Asp Tyr Trp Lys Asn Ala Ala Phe Leu Tyr Gly Leu Gly Leu Val
125 130 135 125 130 135
tac ttc cat tat aat gca ttt cag tgg gca att aaa gca ttt cag gag 842tac ttc cat tat aat gca ttt cag tgg gca att aaa gca ttt cag gag 842
Tyr Phe His Tyr Asn Ala Phe Gln Trp Ala Ile Lys Ala Phe Gln GluTyr Phe His Tyr Asn Ala Phe Gln Trp Ala Ile Lys Ala Phe Gln Glu
140 145 150 155140 145 150 155
gtg ctt tat gtt gat ccc agc ttt tgt cga gcc aag gaa att cat tta 890gtg ctt tat gtt gat ccc agc ttt tgt cga gcc aag gaa att cat tta 890
Val Leu Tyr Val Asp Pro Ser Phe Cys Arg Ala Lys Glu Ile His LeuVal Leu Tyr Val Asp Pro Ser Phe Cys Arg Ala Lys Glu Ile His Leu
160 165 170 160 165 170
cga ctt ggg ctt atg ttc aaa gtg aac aca gac tat gag tct agt tta 938cga ctt ggg ctt atg ttc aaa gtg aac aca gac tat gag tct agt tta 938
Arg Leu Gly Leu Met Phe Lys Val Asn Thr Asp Tyr Glu Ser Ser LeuArg Leu Gly Leu Met Phe Lys Val Asn Thr Asp Tyr Glu Ser Ser Leu
175 180 185 175 180 185
aag cat ttt cag tta gct ttg gtt gac tgt aat ccc tgc act ttg tcc 986aag cat ttt cag tta gct ttg gtt gac tgt aat ccc tgc act ttg tcc 986
Lys His Phe Gln Leu Ala Leu Val Asp Cys Asn Pro Cys Thr Leu SerLys His Phe Gln Leu Ala Leu Val Asp Cys Asn Pro Cys Thr Leu Ser
190 195 200 190 195 200
aat gct gaa att caa ttt cac att gcc cac tta tat gaa acc cag agg 1034aat gct gaa att caa ttt cac att gcc cac tta tat gaa acc cag agg 1034
Asn Ala Glu Ile Gln Phe His Ile Ala His Leu Tyr Glu Thr Gln ArgAsn Ala Glu Ile Gln Phe His Ile Ala His Leu Tyr Glu Thr Gln Arg
205 210 215 205 210 215
aaa tat cat tct gca aaa gaa gct tat gaa caa ctt ttg cag aca gag 1082aaa tat cat tct gca aaa gaa gct tat gaa caa ctt ttg cag aca gag 1082
Lys Tyr His Ser Ala Lys Glu Ala Tyr Glu Gln Leu Leu Gln Thr GluLys Tyr His Ser Ala Lys Glu Ala Tyr Glu Gln Leu Leu Gln Thr Glu
220 225 230 235220 225 230 235
aat ctt tct gca caa gta aaa gca act gtc tta caa cag tta ggt tgg 1130aat ctt tct gca caa gta aaa gca act gtc tta caa cag tta ggt tgg 1130
Asn Leu Ser Ala Gln Val Lys Ala Thr Val Leu Gln Gln Leu Gly TrpAsn Leu Ser Ala Gln Val Lys Ala Thr Val Leu Gln Gln Leu Gly Trp
240 245 250 240 245 250
atg cat cac act gta gat ctc ctg gga gat aaa gcc acc aag gaa agc 1178atg cat cac act gta gat ctc ctg gga gat aaa gcc acc aag gaa agc 1178
Met His His Thr Val Asp Leu Leu Gly Asp Lys Ala Thr Lys Glu SerMet His His Thr Val Asp Leu Leu Gly Asp Lys Ala Thr Lys Glu Ser
255 260 265 255 260 265
tat gct att cag tat ctc caa aag tcc ttg gaa gca gat cct aat tct 1226tat gct att cag tat ctc caa aag tcc ttg gaa gca gat cct aat tct 1226
Tyr Ala Ile Gln Tyr Leu Gln Lys Ser Leu Glu Ala Asp Pro Asn SerTyr Ala Ile Gln Tyr Leu Gln Lys Ser Leu Glu Ala Asp Pro Asn Ser
270 275 280 270 275 280
ggc cag tcc tgg tat ttc ctc gga agg tgc tat tca agt att ggg aaa 1274ggc cag tcc tgg tat ttc ctc gga agg tgc tat tca agt att ggg aaa 1274
Gly Gln Ser Trp Tyr Phe Leu Gly Arg Cys Tyr Ser Ser Ile Gly LysGly Gln Ser Trp Tyr Phe Leu Gly Arg Cys Tyr Ser Ser Ile Gly Lys
285 290 295 285 290 295
gtt cag gat gcc ttt ata tct tac agg cag tct att gat aaa tca gaa 1322gtt cag gat gcc ttt ata tct tac agg cag tct att gat aaa tca gaa 1322
Val Gln Asp Ala Phe Ile Ser Tyr Arg Gln Ser Ile Asp Lys Ser GluVal Gln Asp Ala Phe Ile Ser Tyr Arg Gln Ser Ile Asp Lys Ser Glu
300 305 310 315300 305 310 315
gca agt gca gat aca tgg tgt tca ata ggt gtg cta tat cag cag caa 1370gca agt gca gat aca tgg tgt tca ata ggt gtg cta tat cag cag caa 1370
Ala Ser Ala Asp Thr Trp Cys Ser Ile Gly Val Leu Tyr Gln Gln GlnAla Ser Ala Asp Thr Trp Cys Ser Ile Gly Val Leu Tyr Gln Gln Gln
320 325 330 320 325 330
aat cag ccc atg gat gct tta cag gcc tat att tgt gct gta caa ttg 1418aat cag ccc atg gat gct tta cag gcc tat att tgt gct gta caa ttg 1418
Asn Gln Pro Met Asp Ala Leu Gln Ala Tyr Ile Cys Ala Val Gln LeuAsn Gln Pro Met Asp Ala Leu Gln Ala Tyr Ile Cys Ala Val Gln Leu
335 340 345 335 340 345
gac cat ggc cat gct gca gcc tgg atg gac cta ggc act ctc tat gaa 1466gac cat ggc cat gct gca gcc tgg atg gac cta ggc act ctc tat gaa 1466
Asp His Gly His Ala Ala Ala Trp Met Asp Leu Gly Thr Leu Tyr GluAsp His Gly His Ala Ala Ala Trp Met Asp Leu Gly Thr Leu Tyr Glu
350 355 360 350 355 360
tcc tgc aac cag cct cag gat gcc att aaa tgc tac tta aat gca act 1514tcc tgc aac cag cct cag gat gcc att aaa tgc tac tta aat gca act 1514
Ser Cys Asn Gln Pro Gln Asp Ala Ile Lys Cys Tyr Leu Asn Ala ThrSer Cys Asn Gln Pro Gln Asp Ala Ile Lys Cys Tyr Leu Asn Ala Thr
365 370 375 365 370 375
aga agc aaa agt tgt agt aat acc tct gca ctt gca gca cga att aag 1562aga agc aaa agt tgt agt aat acc tct gca ctt gca gca cga att aag 1562
Arg Ser Lys Ser Cys Ser Asn Thr Ser Ala Leu Ala Ala Arg Ile LysArg Ser Lys Ser Cys Ser Asn Thr Ser Ala Leu Ala Ala Arg Ile Lys
380 385 390 395380 385 390 395
tat tta cag gct cag ttg tgt aac ctt cca caa ggt agt cta cag aat 1610tat tta cag gct cag ttg tgt aac ctt cca caa ggt agt cta cag aat 1610
Tyr Leu Gln Ala Gln Leu Cys Asn Leu Pro Gln Gly Ser Leu Gln AsnTyr Leu Gln Ala Gln Leu Cys Asn Leu Pro Gln Gly Ser Leu Gln Asn
400 405 410 400 405 410
aaa act aaa tta ctt cct agt att gag gag gcg tgg agc cta cca att 1658aaa act aaa tta ctt cct agt att gag gag gcg tgg agc cta cca att 1658
Lys Thr Lys Leu Leu Pro Ser Ile Glu Glu Ala Trp Ser Leu Pro IleLys Thr Lys Leu Leu Pro Ser Ile Glu Glu Ala Trp Ser Leu Pro Ile
415 420 425 415 420 425
ccc gca gag ctt acc tcc agg cag ggt gcc atg aac aca gca cag cag 1706ccc gca gag ctt acc tcc agg cag ggt gcc atg aac aca gca cag cag 1706
Pro Ala Glu Leu Thr Ser Arg Gln Gly Ala Met Asn Thr Ala Gln GlnPro Ala Glu Leu Thr Ser Arg Gln Gly Ala Met Asn Thr Ala Gln Gln
430 435 440 430 435 440
aat act tct gac aat tgg agt ggt gga cat gct gtg tca cat cct cca 1754aat act tct gac aat tgg agt ggt gga cat gct gtg tca cat cct cca 1754
Asn Thr Ser Asp Asn Trp Ser Gly Gly His Ala Val Ser His Pro ProAsn Thr Ser Asp Asn Trp Ser Gly Gly His Ala Val Ser His Pro Pro
445 450 455 445 450 455
gta cag caa caa gct cat tca tgg tgt ttg aca cca cag aaa tta cag 1802gta cag caa caa gct cat tca tgg tgt ttg aca cca cag aaa tta cag 1802
Val Gln Gln Gln Ala His Ser Trp Cys Leu Thr Pro Gln Lys Leu GlnVal Gln Gln Gln Ala His Ser Trp Cys Leu Thr Pro Gln Lys Leu Gln
460 465 470 475460 465 470 475
cat ttg gaa cag ctc cgc gca aat aga aat aat tta aat cca gca cag 1850cat ttg gaa cag ctc cgc gca aat aga aat aat tta aat cca gca cag 1850
His Leu Glu Gln Leu Arg Ala Asn Arg Asn Asn Leu Asn Pro Ala GlnHis Leu Glu Gln Leu Arg Ala Asn Arg Asn Asn Leu Asn Pro Ala Gln
480 485 490 480 485 490
aaa ctg atg ctg gaa cag ctg gaa agt cag ttt gtc tta atg caa caa 1898aaa ctg atg ctg gaa cag ctg gaa agt cag ttt gtc tta atg caa caa 1898
Lys Leu Met Leu Glu Gln Leu Glu Ser Gln Phe Val Leu Met Gln GlnLys Leu Met Leu Glu Gln Leu Glu Ser Gln Phe Val Leu Met Gln Gln
495 500 505 495 500 505
cac caa atg aga cca aca gga gtt gca cag gta cga tct act gga att 1946cac caa atg aga cca aca gga gtt gca cag gta cga tct act gga att 1946
His Gln Met Arg Pro Thr Gly Val Ala Gln Val Arg Ser Thr Gly IleHis Gln Met Arg Pro Thr Gly Val Ala Gln Val Arg Ser Thr Gly Ile
510 515 520 510 515 520
cct aat ggg cca aca gct gac tca tca ctg cct aca aac tca gtc tct 1994cct aat ggg cca aca gct gac tca tca ctg cct aca aac tca gtc tct 1994
Pro Asn Gly Pro Thr Ala Asp Ser Ser Leu Pro Thr Asn Ser Val SerPro Asn Gly Pro Thr Ala Asp Ser Ser Leu Pro Thr Asn Ser Val Ser
525 530 535 525 530 535
ggc cag cag cca cag ctt gct ctg acc aga gtg cct agc gtc tct cag 2042ggc cag cag cca cag ctt gct ctg acc aga gtg cct agc gtc tct cag 2042
Gly Gln Gln Pro Gln Leu Ala Leu Thr Arg Val Pro Ser Val Ser GlnGly Gln Gln Pro Gln Leu Ala Leu Thr Arg Val Pro Ser Val Ser Gln
540 545 550 555540 545 550 555
cct gga gtc cgt cct gcc tgc cct ggg cag cct ttg gcc aat gga ccc 2090cct gga gtc cgt cct gcc tgc cct ggg cag cct ttg gcc aat gga ccc 2090
Pro Gly Val Arg Pro Ala Cys Pro Gly Gln Pro Leu Ala Asn Gly ProPro Gly Val Arg Pro Ala Cys Pro Gly Gln Pro Leu Ala Asn Gly Pro
560 565 570 560 565 570
ttt tct gca ggc cat gtt ccc tgt agc aca tca aga acg ctg gga agt 2138ttt tct gca ggc cat gtt ccc tgt agc aca tca aga acg ctg gga agt 2138
Phe Ser Ala Gly His Val Pro Cys Ser Thr Ser Arg Thr Leu Gly SerPhe Ser Ala Gly His Val Pro Cys Ser Thr Ser Arg Thr Leu Gly Ser
575 580 585 575 580 585
aca gac act att ttg ata ggc aat aat cat ata aca gga agt gga agt 2186aca gac act att ttg ata ggc aat aat cat ata aca gga agt gga agt 2186
Thr Asp Thr Ile Leu Ile Gly Asn Asn His Ile Thr Gly Ser Gly SerThr Asp Thr Ile Leu Ile Gly Asn Asn His Ile Thr Gly Ser Gly Ser
590 595 600 590 595 600
aat gga aac gtg cct tac ctg cag cga aac gca ctc act cta cct cat 2234aat gga aac gtg cct tac ctg cag cga aac gca ctc act cta cct cat 2234
Asn Gly Asn Val Pro Tyr Leu Gln Arg Asn Ala Leu Thr Leu Pro HisAsn Gly Asn Val Pro Tyr Leu Gln Arg Asn Ala Leu Thr Leu Pro His
605 610 615 605 610 615
aac cgc aca aac ctg acc agc agc gca gag gag ccg tgg aaa aac caa 2282aac cgc aca aac ctg acc agc agc gca gag gag ccg tgg aaa aac caa 2282
Asn Arg Thr Asn Leu Thr Ser Ser Ala Glu Glu Pro Trp Lys Asn GlnAsn Arg Thr Asn Leu Thr Ser Ser Ala Glu Glu Pro Trp Lys Asn Gln
620 625 630 635620 625 630 635
cta tct aac tcc act cag ggg ctt cac aaa ggt cag agt tca cat tcg 2330cta tct aac tcc act cag ggg ctt cac aaa ggt cag agt tca cat tcg 2330
Leu Ser Asn Ser Thr Gln Gly Leu His Lys Gly Gln Ser Ser His SerLeu Ser Asn Ser Thr Gln Gly Leu His Lys Gly Gln Ser Ser His Ser
640 645 650 640 645 650
gca ggt cct aat ggt gaa cga cct ctc tct tcc act ggg cct tcc cag 2378gca ggt cct aat ggt gaa cga cct ctc tct tcc act ggg cct tcc cag 2378
Ala Gly Pro Asn Gly Glu Arg Pro Leu Ser Ser Thr Gly Pro Ser GlnAla Gly Pro Asn Gly Glu Arg Pro Leu Ser Ser Thr Gly Pro Ser Gln
655 660 665 655 660 665
cat ctc cag gca gct ggc tct ggt att cag aat cag aac gga cat ccc 2426cat ctc cag gca gct ggc tct ggt att cag aat cag aac gga cat ccc 2426
His Leu Gln Ala Ala Gly Ser Gly Ile Gln Asn Gln Asn Gly His ProHis Leu Gln Ala Ala Gly Ser Gly Ile Gln Asn Gln Asn Gly His Pro
670 675 680 670 675 680
acc ctg cct agc aat tca gta aca cag ggg gct gct ctc aat cac ctc 2474acc ctg cct agc aat tca gta aca cag ggg gct gct ctc aat cac ctc 2474
Thr Leu Pro Ser Asn Ser Val Thr Gln Gly Ala Ala Leu Asn His LeuThr Leu Pro Ser Asn Ser Val Thr Gln Gly Ala Ala Leu Asn His Leu
685 690 695 685 690 695
tcc tct cac act gct acc tca ggt gga caa caa ggc att acc tta acc 2522tcc tct cac act gct acc tca ggt gga caa caa ggc att acc tta acc 2522
Ser Ser His Thr Ala Thr Ser Gly Gly Gln Gln Gly Ile Thr Leu ThrSer Ser His Thr Ala Thr Ser Gly Gly Gln Gln Gly Ile Thr Leu Thr
700 705 710 715700 705 710 715
aaa gag agc aag cct tca gga aac ata ttg acg gtg cct gaa aca agc 2570aaa gag agc aag cct tca gga aac ata ttg acg gtg cct gaa aca agc 2570
Lys Glu Ser Lys Pro Ser Gly Asn Ile Leu Thr Val Pro Glu Thr SerLys Glu Ser Lys Pro Ser Gly Asn Ile Leu Thr Val Pro Glu Thr Ser
720 725 730 720 725 730
agg cac act gga gag aca cct aac agc act gcc agt gtc gag gga ctt 2618agg cac act gga gag aca cct aac agc act gcc agt gtc gag gga ctt 2618
Arg His Thr Gly Glu Thr Pro Asn Ser Thr Ala Ser Val Glu Gly LeuArg His Thr Gly Glu Thr Pro Asn Ser Thr Ala Ser Val Glu Gly Leu
735 740 745 735 740 745
cct aat cat gtc cat cag atg acg gca gat gct gtt tgc agt cct agc 2666cct aat cat gtc cat cag atg acg gca gat gct gtt tgc agt cct agc 2666
Pro Asn His Val His Gln Met Thr Ala Asp Ala Val Cys Ser Pro SerPro Asn His Val His Gln Met Thr Ala Asp Ala Val Cys Ser Pro Ser
750 755 760 750 755 760
cat gga gat tct aag tca cca ggt tta cta agt tca gac aat cct cag 2714cat gga gat tct aag tca cca ggt tta cta agt tca gac aat cct cag 2714
His Gly Asp Ser Lys Ser Pro Gly Leu Leu Ser Ser Asp Asn Pro GlnHis Gly Asp Ser Lys Ser Pro Gly Leu Leu Ser Ser Asp Asn Pro Gln
765 770 775 765 770 775
ctc tct gcc ttg ttg atg gga aaa gcc aat aac aat gtg ggt act gga 2762ctc tct gcc ttg ttg atg gga aaa gcc aat aac aat gtg ggt act gga 2762
Leu Ser Ala Leu Leu Met Gly Lys Ala Asn Asn Asn Val Gly Thr GlyLeu Ser Ala Leu Leu Met Gly Lys Ala Asn Asn Asn Val Gly Thr Gly
780 785 790 795780 785 790 795
acc tgt gac aaa gtc aat aac atc cac cca gct gtt cat aca aag act 2810acc tgt gac aaa gtc aat aac atc cac cca gct gtt cat aca aag act 2810
Thr Cys Asp Lys Val Asn Asn Ile His Pro Ala Val His Thr Lys ThrThr Cys Asp Lys Val Asn Asn Ile His Pro Ala Val His Thr Lys Thr
800 805 810 800 805 810
gat aac tct gtt gcc tct tca cca tct tca gcc att tca aca gca aca 2858gat aac tct gtt gcc tct tca cca tct tca gcc att tca aca gca aca 2858
Asp Asn Ser Val Ala Ser Ser Pro Ser Ser Ala Ile Ser Thr Ala ThrAsp Asn Ser Val Ala Ser Ser Pro Ser Ser Ala Ile Ser Thr Ala Thr
815 820 825 815 820 825
cct tct cca aaa tcc act gag cag aca acc aca aac agt gtt acc agc 2906cct tct cca aaa tcc act gag cag aca acc aca aac agt gtt acc agc 2906
Pro Ser Pro Lys Ser Thr Glu Gln Thr Thr Thr Asn Ser Val Thr SerPro Ser Pro Lys Ser Thr Glu Gln Thr Thr Thr Asn Ser Val Thr Ser
830 835 840 830 835 840
ctt aac agc cct cac agt ggg cta cac aca att aat gga gaa ggg atg 2954ctt aac agc cct cac agt ggg cta cac aca att aat gga gaa ggg atg 2954
Leu Asn Ser Pro His Ser Gly Leu His Thr Ile Asn Gly Glu Gly MetLeu Asn Ser Pro His Ser Gly Leu His Thr Ile Asn Gly Glu Gly Met
845 850 855 845 850 855
gaa gaa tct cag agc ccc atg aaa aca gat ctg ctt ctg gtt aac cac 3002gaa gaa tct cag agc ccc atg aaa aca gat ctg ctt ctg gtt aac cac 3002
Glu Glu Ser Gln Ser Pro Met Lys Thr Asp Leu Leu Leu Val Asn HisGlu Glu Ser Gln Ser Pro Met Lys Thr Asp Leu Leu Leu Val Asn His
860 865 870 875860 865 870 875
aaa cct agt cca cag atc ata cca tca atg tct gtg tcc ata tac ccc 3050aaa cct agt cca cag atc ata cca tca atg tct gtg tcc ata tac ccc 3050
Lys Pro Ser Pro Gln Ile Ile Pro Ser Met Ser Val Ser Ile Tyr ProLys Pro Ser Pro Gln Ile Ile Pro Ser Met Ser Val Ser Ile Tyr Pro
880 885 890 880 885 890
agc tca gca gaa gtt ctg aag gca tgc agg aat cta ggt aaa aat ggc 3098agc tca gca gaa gtt ctg aag gca tgc agg aat cta ggt aaa aat ggc 3098
Ser Ser Ala Glu Val Leu Lys Ala Cys Arg Asn Leu Gly Lys Asn GlySer Ser Ala Glu Val Leu Lys Ala Cys Arg Asn Leu Gly Lys Asn Gly
895 900 905 895 900 905
tta tct aac agt agc att ttg ttg gat aaa tgt cca cct cca aga cca 3146tta tct aac agt agc att ttg ttg gat aaa tgt cca cct cca aga cca 3146
Leu Ser Asn Ser Ser Ile Leu Leu Asp Lys Cys Pro Pro Pro Arg ProLeu Ser Asn Ser Ser Ile Leu Leu Asp Lys Cys Pro Pro Pro Pro Arg Pro
910 915 920 910 915 920
cca tct tca cca tac cct ccc ttg cca aag gac aag ttg aat cca cct 3194cca tct tca cca tac cct ccc ttg cca aag gac aag ttg aat cca cct 3194
Pro Ser Ser Pro Tyr Pro Pro Leu Pro Lys Asp Lys Leu Asn Pro ProPro Ser Ser Pro Tyr Pro Pro Leu Pro Lys Asp Lys Leu Asn Pro Pro
925 930 935 925 930 935
aca cct agt att tac ttg gaa aat aaa cgt gat gct ttc ttt cct cca 3242aca cct agt att tac ttg gaa aat aaa cgt gat gct ttc ttt cct cca 3242
Thr Pro Ser Ile Tyr Leu Glu Asn Lys Arg Asp Ala Phe Phe Pro ProThr Pro Ser Ile Tyr Leu Glu Asn Lys Arg Asp Ala Phe Phe Pro Pro
940 945 950 955940 945 950 955
tta cat caa ttt tgt aca aat ccg aac aac cct gtt aca gta ata cgt 3290tta cat caa ttt tgt aca aat ccg aac aac cct gtt aca gta ata cgt 3290
Leu His Gln Phe Cys Thr Asn Pro Asn Asn Pro Val Thr Val Ile ArgLeu His Gln Phe Cys Thr Asn Pro Asn Asn Pro Val Thr Val Ile Arg
960 965 970 960 965 970
ggc ctt gct gga gct ctt aag tta gac ctg gga ctt ttc tct act aaa 3338ggc ctt gct gga gct ctt aag tta gac ctg gga ctt ttc tct act aaa 3338
Gly Leu Ala Gly Ala Leu Lys Leu Asp Leu Gly Leu Phe Ser Thr LysGly Leu Ala Gly Ala Leu Lys Leu Asp Leu Gly Leu Phe Ser Thr Lys
975 980 985 975 980 985
act ttg gtg gaa gct aac aat gaa cat atg gta gaa gtg agg aca cag 3386act ttg gtg gaa gct aac aat gaa cat atg gta gaa gtg agg aca cag 3386
Thr Leu Val Glu Ala Asn Asn Glu His Met Val Glu Val Arg Thr GlnThr Leu Val Glu Ala Asn Asn Glu His Met Val Glu Val Arg Thr Gln
990 995 1000 990 995 1000
ttg ttg cag cca gca gat gaa aac tgg gat ccc act gga aca aag 3431ttg ttg cag cca gca gat gaa aac tgg gat ccc act gga aca aag 3431
Leu Leu Gln Pro Ala Asp Glu Asn Trp Asp Pro Thr Gly Thr LysLeu Leu Gln Pro Ala Asp Glu Asn Trp Asp Pro Thr Gly Thr Lys
1005 1010 1015 1005 1010 1015
aaa atc tgg cat tgt gaa agt aat aga tct cat act aca att gct 3476aaa atc tgg cat tgt gaa agt aat aga tct cat act aca att gct 3476
Lys Ile Trp His Cys Glu Ser Asn Arg Ser His Thr Thr Ile AlaLys Ile Trp His Cys Glu Ser Asn Arg Ser His Thr Thr Ile Ala
1020 1025 1030 1020 1025 1030
aaa tat gca cag tac cag gcc tcc tca ttc cag gaa tca ttg aga 3521aaa tat gca cag tac cag gcc tcc tca ttc cag gaa tca ttg aga 3521
Lys Tyr Ala Gln Tyr Gln Ala Ser Ser Phe Gln Glu Ser Leu ArgLys Tyr Ala Gln Tyr Gln Ala Ser Ser Phe Gln Glu Ser Leu Arg
1035 1040 1045 1035 1040 1045
gaa gaa aat gaa aaa aga agt cat cat aaa gac cac tca gat agt 3566gaa gaa aat gaa aaa aga agt cat cat aaa gac cac tca gat agt 3566
Glu Glu Asn Glu Lys Arg Ser His His Lys Asp His Ser Asp SerGlu Glu Asn Glu Lys Arg Ser His His Lys Asp His Ser Asp Ser
1050 1055 1060 1050 1055 1060
gaa tct aca tcg tca gat aat tct ggg agg agg agg aaa gga ccc 3611gaa tct aca tcg tca gat aat tct ggg agg agg agg aaa gga ccc 3611
Glu Ser Thr Ser Ser Asp Asn Ser Gly Arg Arg Arg Lys Gly ProGlu Ser Thr Ser Ser Asp Asn Ser Gly Arg Arg Arg Lys Gly Pro
1065 1070 1075 1065 1070 1075
ttt aaa acc ata aag ttt ggg acc aat att gac cta tct gat gac 3656ttt aaa acc ata aag ttt ggg acc aat att gac cta tct gat gac 3656
Phe Lys Thr Ile Lys Phe Gly Thr Asn Ile Asp Leu Ser Asp AspPhe Lys Thr Ile Lys Phe Gly Thr Asn Ile Asp Leu Ser Asp Asp
1080 1085 1090 1080 1085 1090
aaa aag tgg aag ttg cag cta cat gag ctg act aaa ctt cct gct 3701aaa aag tgg aag ttg cag cta cat gag ctg act aaa ctt cct gct 3701
Lys Lys Trp Lys Leu Gln Leu His Glu Leu Thr Lys Leu Pro AlaLys Lys Trp Lys Leu Gln Leu His Glu Leu Thr Lys Leu Pro Ala
1095 1100 1105 1095 1100 1105
ttt gtg cgt gtc gta tca gca gga aat ctt cta agc cat gtt ggt 3746ttt gtg cgt gtc gta tca gca gga aat ctt cta agc cat gtt ggt 3746
Phe Val Arg Val Val Ser Ala Gly Asn Leu Leu Ser His Val GlyPhe Val Arg Val Val Ser Ala Gly Asn Leu Leu Ser His Val Gly
1110 1115 1120 1110 1115 1120
cat acc ata ttg ggc atg aac aca gtt caa cta tac atg aaa gtt 3791cat acc ata ttg ggc atg aac aca gtt caa cta tac atg aaa gtt 3791
His Thr Ile Leu Gly Met Asn Thr Val Gln Leu Tyr Met Lys ValHis Thr Ile Leu Gly Met Asn Thr Val Gln Leu Tyr Met Lys Val
1125 1130 1135 1125 1130 1135
cca ggg agc aga aca cca ggt cat cag gaa aat aac aac ttc tgt 3836cca ggg agc aga aca cca ggt cat cag gaa aat aac aac ttc tgt 3836
Pro Gly Ser Arg Thr Pro Gly His Gln Glu Asn Asn Asn Phe CysPro Gly Ser Arg Thr Pro Gly His Gln Glu Asn Asn Asn Asn Phe Cys
1140 1145 1150 1140 1145 1150
tca gtt aac ata aat att ggc cca ggt gac tgt gaa tgg ttt gtt 3881tca gtt aac ata aat att ggc cca ggt gac tgt gaa tgg ttt gtt 3881
Ser Val Asn Ile Asn Ile Gly Pro Gly Asp Cys Glu Trp Phe ValSer Val Asn Ile Asn Ile Gly Pro Gly Asp Cys Glu Trp Phe Val
1155 1160 1165 1155 1160 1165
gtt cct gaa ggt tac tgg ggt gtt ctg aat gac ttc tgt gaa aaa 3926gtt cct gaa ggt tac tgg ggt gtt ctg aat gac ttc tgt gaa aaa 3926
Val Pro Glu Gly Tyr Trp Gly Val Leu Asn Asp Phe Cys Glu LysVal Pro Glu Gly Tyr Trp Gly Val Leu Asn Asp Phe Cys Glu Lys
1170 1175 1180 1170 1175 1180
aat aat ttg aat ttc cta atg ggt tct tgg tgg ccc aat ctt gaa 3971aat aat ttg aat ttc cta atg ggt tct tgg tgg ccc aat ctt gaa 3971
Asn Asn Leu Asn Phe Leu Met Gly Ser Trp Trp Pro Asn Leu GluAsn Asn Leu Asn Phe Leu Met Gly Ser Trp Trp Pro Asn Leu Glu
1185 1190 1195 1185 1190 1195
gat ctt tat gaa gca aat gtt cca gtg tat agg ttt att cag cga 4016gat ctt tat gaa gca aat gtt cca gtg tat agg ttt att cag cga 4016
Asp Leu Tyr Glu Ala Asn Val Pro Val Tyr Arg Phe Ile Gln ArgAsp Leu Tyr Glu Ala Asn Val Pro Val Tyr Arg Phe Ile Gln Arg
1200 1205 1210 1200 1205 1210
cct gga gat ttg gtc tgg ata aat gca ggc act gtt cat tgg gtt 4061cct gga gat ttg gtc tgg aat aat gca ggc act gtt cat tgg gtt 4061
Pro Gly Asp Leu Val Trp Ile Asn Ala Gly Thr Val His Trp ValPro Gly Asp Leu Val Trp Ile Asn Ala Gly Thr Val His Trp Val
1215 1220 1225 1215 1220 1225
cag gct att ggc tgg tgc aac aac att gct tgg aat gtt ggt cca 4106cag gct att ggc tgg tgc aac aac att gct tgg aat gtt ggt cca 4106
Gln Ala Ile Gly Trp Cys Asn Asn Ile Ala Trp Asn Val Gly ProGln Ala Ile Gly Trp Cys Asn Asn Ile Ala Trp Asn Val Gly Pro
1230 1235 1240 1230 1235 1240
ctt aca gcc tgc cag tat aaa ttg gca gtg gaa cgg tac gaa tgg 4151ctt aca gcc tgc cag tat aaa ttg gca gtg gaa cgg tac gaa tgg 4151
Leu Thr Ala Cys Gln Tyr Lys Leu Ala Val Glu Arg Tyr Glu TrpLeu Thr Ala Cys Gln Tyr Lys Leu Ala Val Glu Arg Tyr Glu Trp
1245 1250 1255 1245 1250 1255
aac aaa ttg caa agt gtg aag tca ata gta ccc atg gtt cat ctt 4196aac aaa ttg caa agt gtg aag tca ata gta ccc atg gtt cat ctt 4196
Asn Lys Leu Gln Ser Val Lys Ser Ile Val Pro Met Val His LeuAsn Lys Leu Gln Ser Val Lys Ser Ile Val Pro Met Val His Leu
1260 1265 1270 1260 1265 1270
tcc tgg aat atg gca cga aat atc aag gtc tca gat cca aag ctt 4241tcc tgg aat atg gca cga aat atc aag gtc tca gat cca aag ctt 4241
Ser Trp Asn Met Ala Arg Asn Ile Lys Val Ser Asp Pro Lys LeuSer Trp Asn Met Ala Arg Asn Ile Lys Val Ser Asp Pro Lys Leu
1275 1280 1285 1275 1280 1285
ttt gaa atg att aag tat tgt ctt cta aga act ctg aag caa tgt 4286ttt gaa atg att aag tat tgt ctt cta aga act ctg aag caa tgt 4286
Phe Glu Met Ile Lys Tyr Cys Leu Leu Arg Thr Leu Lys Gln CysPhe Glu Met Ile Lys Tyr Cys Leu Leu Arg Thr Leu Lys Gln Cys
1290 1295 1300 1290 1295 1300
cag aca ttg agg gaa gct ctc att gct gca gga aaa gag att ata 4331cag aca ttg agg gaa gct ctc att gct gca gga aaa gag att ata 4331
Gln Thr Leu Arg Glu Ala Leu Ile Ala Ala Gly Lys Glu Ile IleGln Thr Leu Arg Glu Ala Leu Ile Ala Ala Gly Lys Glu Ile Ile
1305 1310 1315 1305 1310 1315
tgg cat ggg cgg aca aaa gaa gaa cca gct cat tac tgt agc att 4376tgg cat ggg cgg aca aaa gaa gaa cca gct cat tac tgt agc att 4376
Trp His Gly Arg Thr Lys Glu Glu Pro Ala His Tyr Cys Ser IleTrp His Gly Arg Thr Lys Glu Glu Pro Ala His Tyr Cys Ser Ile
1320 1325 1330 1320 1325 1330
tgt gaa gtg gag gtt ttt gat ctg ctt ttt gtc act aat gag agt 4421tgt gaa gtg gag gtt ttt gat ctg ctt ttt gtc act aat gag agt 4421
Cys Glu Val Glu Val Phe Asp Leu Leu Phe Val Thr Asn Glu SerCys Glu Val Glu Val Phe Asp Leu Leu Phe Val Thr Asn Glu Ser
1335 1340 1345 1335 1340 1345
aat tca cga aag acc tac ata gta cat tgc caa gat tgt gca cga 4466aat tca cga aag acc tac ata gta cat tgc caa gat tgt gca cga 4466
Asn Ser Arg Lys Thr Tyr Ile Val His Cys Gln Asp Cys Ala ArgAsn Ser Arg Lys Thr Tyr Ile Val His Cys Gln Asp Cys Ala Arg
1350 1355 1360 1350 1355 1360
aaa aca agc gga aac ttg gaa aac ttt gtg gtg cta gaa cag tac 4511aaa aca agc gga aac ttg gaa aac ttt gtg gtg cta gaa cag tac 4511
Lys Thr Ser Gly Asn Leu Glu Asn Phe Val Val Leu Glu Gln TyrLys Thr Ser Gly Asn Leu Glu Asn Phe Val Val Leu Glu Gln Tyr
1365 1370 1375 1365 1370 1375
aaa atg gag gac ctg atg caa gtc tat gac caa ttt aca tta gct 4556aaa atg gag gac ctg atg caa gtc tat gac caa ttt aca tta gct 4556
Lys Met Glu Asp Leu Met Gln Val Tyr Asp Gln Phe Thr Leu AlaLys Met Glu Asp Leu Met Gln Val Tyr Asp Gln Phe Thr Leu Ala
1380 1385 1390 1380 1385 1390
cct cca tta cca tcc gcc tca tct tga tattgttcca tggacattaa 4603cct cca tta cca tcc gcc tca tct tga tattgttcca tggacattaa 4603
Pro Pro Leu Pro Ser Ala Ser SerPro Pro Leu Pro Ser Ala Ser Ser
1395 1400 1395 1400
atgagacctt ttctgctatt caggaaataa cccagttctg caccactggt ttttgtagct 4663atgagacctt ttctgctatt caggaaataa cccagttctg caccactggt ttttgtagct 4663
atctcgtaag gctgctggct gaaaactgtg tctatgcaac cttccaagtg cggagtgtca 4723atctcgtaag gctgctggct gaaaactgtg tctatgcaac cttccaagtg cggagtgtca 4723
accaactgga cgggagagag tactgctcct actccaggac tctcacaaag ctgatgagct 4783accaactgga cggggagagag tactgctcct actccaggac tctcacaaag ctgatgagct 4783
gtacttcaga aaaaaataat aatttccatg ttttgtatat atctgacaaa actggcaaca 4843gtacttcaga aaaaaataat aatttccatg ttttgtatat atctgacaaa actggcaaca 4843
tcttacagac tactgacttg aagacaacct cttttatatt tctctatttc tgggctgatg 4903tcttacagac tactgacttg aagacaacct cttttatatt tctctatttc tgggctgatg 4903
aatttgtttt catctgtctt ttcccccttc agaattttcc ttggaaaaaa aatactagcc 4963aatttgtttt catctgtctt ttcccccttc agaattttcc ttggaaaaaa aatactagcc 4963
tagctggtca tttctttgta aggtagttag caattttaag tctttctttg gtcaactttt 5023tagctggtca tttctttgta aggtagttag caattttaag tctttctttg gtcaactttt 5023
ttttaatgtg aaaagttagg taagacactt ttttactgct tttatgtttt tctgtcttgt 5083ttttaatgtg aaaagttagg taagacactt ttttatactgct tttatgtttt tctgtcttgt 5083
tttgagacca tgatggttac acttttggtt cctaaataaa atttaaaaaa ttaacagcca 5143tttgagacca tgatggttac acttttggtt cctaaataaa atttaaaaaa ttaacagcca 5143
agtcacaaag gtaatggatt gcacatagac taaggaataa acttcagatt tgtgattttt 5203agtcacaaag gtaatggatt gcacatagac taaggaataa acttcagatt tgtgattttt 5203
gtttctaatc ttgatgtaaa tttacactat ttataaatac atatttattg cttgaaaata 5263gtttctaatc ttgatgtaaa tttacactat ttataaatac atatttattg cttgaaaata 5263
tttgtgaatg gaatgctgtt attttttcca gatttacctg ccattgaaat tttaaggagt 5323tttgtgaatg gaatgctgtt attttttcca gatttacctg ccattgaaat tttaaggagt 5323
tctgtaattt caaacactac tcctattaca ttttctatgt gtaaataaaa ctgcttagca 5383tctgtaattt caaacactac tccttattaca ttttctatgt gtaaataaaa ctgcttagca 5383
ttgtacagaa acttttatta aaattgttta atgtttaaag agttttctat tgtttgagtt 5443ttgtacagaa acttttatta aaattgttta atgtttaaag agttttctat tgtttgagtt 5443
ttaaaaaaga ctttatgtac agtgcccagt ttttgttcat ttttgaaatc tgattatata 5503ttaaaaaaga ctttatgtac agtgcccagt ttttgttcat ttttgaaatc tgattatata 5503
tattttatat atacttatgt atgtatatat aatatatata gaaatctgga tatatatgta 5563tattttatat atacttatgt atgtatatat aatatatata gaaatctgga tatatatgta 5563
taaatcttta gaacttaaat ttttctcgtt ttaagtttca catctatggt agatttttga 5623taaatcttta gaacttaaat ttttctcgtt ttaagtttca catctatggt agatttttga 5623
ggtgtctact gtaaagtatt gcttacaaaa agtatgatta tttttaaaga aatatatatg 5683ggtgtctact gtaaagtatt gcttacaaaa agtatgatta tttttaaaga aatatatatg 5683
gtatgtatcc tcaagaccta aaatgtcaga ctggtttatt gttaagttgc aattactgca 5743gtatgtatcc tcaagaccta aaatgtcaga ctggtttatt gttaagttgc aattactgca 5743
atgacagacc aataaacaat tgctgccaaa atgtagtata aa 5785atgacagacc aataaacaat tgctgccaaa atgtagtata aa 5785
<210> 2<210> 2
<211> 1401<211> 1401
<212> PRT<212> PRT
<213> 智人(Homo sapiens)<213> Homo sapiens
<400> 2<400> 2
Met Lys Ser Cys Gly Val Ser Leu Ala Thr Ala Ala Ala Ala Ala AlaMet Lys Ser Cys Gly Val Ser Leu Ala Thr Ala Ala Ala Ala Ala Ala
1 5 10 151 5 10 15
Ala Phe Gly Asp Glu Glu Lys Lys Met Ala Ala Gly Lys Ala Ser GlyAla Phe Gly Asp Glu Glu Lys Lys Met Ala Ala Gly Lys Ala Ser Gly
20 25 30 20 25 30
Glu Ser Glu Glu Ala Ser Pro Ser Leu Thr Ala Glu Glu Arg Glu AlaGlu Ser Glu Glu Ala Ser Pro Ser Leu Thr Ala Glu Glu Arg Glu Ala
35 40 45 35 40 45
Leu Gly Gly Leu Asp Ser Arg Leu Phe Gly Phe Val Arg Phe His GluLeu Gly Gly Leu Asp Ser Arg Leu Phe Gly Phe Val Arg Phe His Glu
50 55 60 50 55 60
Asp Gly Ala Arg Thr Lys Ala Leu Leu Gly Lys Ala Val Arg Cys TyrAsp Gly Ala Arg Thr Lys Ala Leu Leu Gly Lys Ala Val Arg Cys Tyr
65 70 75 8065 70 75 80
Glu Ser Leu Ile Leu Lys Ala Glu Gly Lys Val Glu Ser Asp Phe PheGlu Ser Leu Ile Leu Lys Ala Glu Gly Lys Val Glu Ser Asp Phe Phe
85 90 95 85 90 95
Cys Gln Leu Gly His Phe Asn Leu Leu Leu Glu Asp Tyr Pro Lys AlaCys Gln Leu Gly His Phe Asn Leu Leu Leu Glu Asp Tyr Pro Lys Ala
100 105 110 100 105 110
Leu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu Gln Ser Asp Tyr Trp LysLeu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu Gln Ser Asp Tyr Trp Lys
115 120 125 115 120 125
Asn Ala Ala Phe Leu Tyr Gly Leu Gly Leu Val Tyr Phe His Tyr AsnAsn Ala Ala Phe Leu Tyr Gly Leu Gly Leu Val Tyr Phe His Tyr Asn
130 135 140 130 135 140
Ala Phe Gln Trp Ala Ile Lys Ala Phe Gln Glu Val Leu Tyr Val AspAla Phe Gln Trp Ala Ile Lys Ala Phe Gln Glu Val Leu Tyr Val Asp
145 150 155 160145 150 155 160
Pro Ser Phe Cys Arg Ala Lys Glu Ile His Leu Arg Leu Gly Leu MetPro Ser Phe Cys Arg Ala Lys Glu Ile His Leu Arg Leu Gly Leu Met
165 170 175 165 170 175
Phe Lys Val Asn Thr Asp Tyr Glu Ser Ser Leu Lys His Phe Gln LeuPhe Lys Val Asn Thr Asp Tyr Glu Ser Ser Leu Lys His Phe Gln Leu
180 185 190 180 185 190
Ala Leu Val Asp Cys Asn Pro Cys Thr Leu Ser Asn Ala Glu Ile GlnAla Leu Val Asp Cys Asn Pro Cys Thr Leu Ser Asn Ala Glu Ile Gln
195 200 205 195 200 205
Phe His Ile Ala His Leu Tyr Glu Thr Gln Arg Lys Tyr His Ser AlaPhe His Ile Ala His Leu Tyr Glu Thr Gln Arg Lys Tyr His Ser Ala
210 215 220 210 215 220
Lys Glu Ala Tyr Glu Gln Leu Leu Gln Thr Glu Asn Leu Ser Ala GlnLys Glu Ala Tyr Glu Gln Leu Leu Gln Thr Glu Asn Leu Ser Ala Gln
225 230 235 240225 230 235 240
Val Lys Ala Thr Val Leu Gln Gln Leu Gly Trp Met His His Thr ValVal Lys Ala Thr Val Leu Gln Gln Leu Gly Trp Met His His Thr Val
245 250 255 245 250 255
Asp Leu Leu Gly Asp Lys Ala Thr Lys Glu Ser Tyr Ala Ile Gln TyrAsp Leu Leu Gly Asp Lys Ala Thr Lys Glu Ser Tyr Ala Ile Gln Tyr
260 265 270 260 265 270
Leu Gln Lys Ser Leu Glu Ala Asp Pro Asn Ser Gly Gln Ser Trp TyrLeu Gln Lys Ser Leu Glu Ala Asp Pro Asn Ser Gly Gln Ser Trp Tyr
275 280 285 275 280 285
Phe Leu Gly Arg Cys Tyr Ser Ser Ile Gly Lys Val Gln Asp Ala PhePhe Leu Gly Arg Cys Tyr Ser Ser Ile Gly Lys Val Gln Asp Ala Phe
290 295 300 290 295 300
Ile Ser Tyr Arg Gln Ser Ile Asp Lys Ser Glu Ala Ser Ala Asp ThrIle Ser Tyr Arg Gln Ser Ile Asp Lys Ser Glu Ala Ser Ala Asp Thr
305 310 315 320305 310 315 320
Trp Cys Ser Ile Gly Val Leu Tyr Gln Gln Gln Asn Gln Pro Met AspTrp Cys Ser Ile Gly Val Leu Tyr Gln Gln Gln Asn Gln Pro Met Asp
325 330 335 325 330 335
Ala Leu Gln Ala Tyr Ile Cys Ala Val Gln Leu Asp His Gly His AlaAla Leu Gln Ala Tyr Ile Cys Ala Val Gln Leu Asp His Gly His Ala
340 345 350 340 345 350
Ala Ala Trp Met Asp Leu Gly Thr Leu Tyr Glu Ser Cys Asn Gln ProAla Ala Trp Met Asp Leu Gly Thr Leu Tyr Glu Ser Cys Asn Gln Pro
355 360 365 355 360 365
Gln Asp Ala Ile Lys Cys Tyr Leu Asn Ala Thr Arg Ser Lys Ser CysGln Asp Ala Ile Lys Cys Tyr Leu Asn Ala Thr Arg Ser Lys Ser Cys
370 375 380 370 375 380
Ser Asn Thr Ser Ala Leu Ala Ala Arg Ile Lys Tyr Leu Gln Ala GlnSer Asn Thr Ser Ala Leu Ala Ala Arg Ile Lys Tyr Leu Gln Ala Gln
385 390 395 400385 390 395 400
Leu Cys Asn Leu Pro Gln Gly Ser Leu Gln Asn Lys Thr Lys Leu LeuLeu Cys Asn Leu Pro Gln Gly Ser Leu Gln Asn Lys Thr Lys Leu Leu
405 410 415 405 410 415
Pro Ser Ile Glu Glu Ala Trp Ser Leu Pro Ile Pro Ala Glu Leu ThrPro Ser Ile Glu Glu Ala Trp Ser Leu Pro Ile Pro Ala Glu Leu Thr
420 425 430 420 425 430
Ser Arg Gln Gly Ala Met Asn Thr Ala Gln Gln Asn Thr Ser Asp AsnSer Arg Gln Gly Ala Met Asn Thr Ala Gln Gln Asn Thr Ser Asp Asn
435 440 445 435 440 445
Trp Ser Gly Gly His Ala Val Ser His Pro Pro Val Gln Gln Gln AlaTrp Ser Gly Gly His Ala Val Ser His Pro Pro Val Gln Gln Gln Ala
450 455 460 450 455 460
His Ser Trp Cys Leu Thr Pro Gln Lys Leu Gln His Leu Glu Gln LeuHis Ser Trp Cys Leu Thr Pro Gln Lys Leu Gln His Leu Glu Gln Leu
465 470 475 480465 470 475 480
Arg Ala Asn Arg Asn Asn Leu Asn Pro Ala Gln Lys Leu Met Leu GluArg Ala Asn Arg Asn Asn Leu Asn Pro Ala Gln Lys Leu Met Leu Glu
485 490 495 485 490 495
Gln Leu Glu Ser Gln Phe Val Leu Met Gln Gln His Gln Met Arg ProGln Leu Glu Ser Gln Phe Val Leu Met Gln Gln His Gln Met Arg Pro
500 505 510 500 505 510
Thr Gly Val Ala Gln Val Arg Ser Thr Gly Ile Pro Asn Gly Pro ThrThr Gly Val Ala Gln Val Arg Ser Thr Gly Ile Pro Asn Gly Pro Thr
515 520 525 515 520 525
Ala Asp Ser Ser Leu Pro Thr Asn Ser Val Ser Gly Gln Gln Pro GlnAla Asp Ser Ser Leu Pro Thr Asn Ser Val Ser Gly Gln Gln Pro Gln
530 535 540 530 535 540
Leu Ala Leu Thr Arg Val Pro Ser Val Ser Gln Pro Gly Val Arg ProLeu Ala Leu Thr Arg Val Pro Ser Val Ser Gln Pro Gly Val Arg Pro
545 550 555 560545 550 555 560
Ala Cys Pro Gly Gln Pro Leu Ala Asn Gly Pro Phe Ser Ala Gly HisAla Cys Pro Gly Gln Pro Leu Ala Asn Gly Pro Phe Ser Ala Gly His
565 570 575 565 570 575
Val Pro Cys Ser Thr Ser Arg Thr Leu Gly Ser Thr Asp Thr Ile LeuVal Pro Cys Ser Thr Ser Arg Thr Leu Gly Ser Thr Asp Thr Ile Leu
580 585 590 580 585 590
Ile Gly Asn Asn His Ile Thr Gly Ser Gly Ser Asn Gly Asn Val ProIle Gly Asn Asn His Ile Thr Gly Ser Gly Ser Asn Gly Asn Val Pro
595 600 605 595 600 605
Tyr Leu Gln Arg Asn Ala Leu Thr Leu Pro His Asn Arg Thr Asn LeuTyr Leu Gln Arg Asn Ala Leu Thr Leu Pro His Asn Arg Thr Asn Leu
610 615 620 610 615 620
Thr Ser Ser Ala Glu Glu Pro Trp Lys Asn Gln Leu Ser Asn Ser ThrThr Ser Ser Ala Glu Glu Pro Trp Lys Asn Gln Leu Ser Asn Ser Thr
625 630 635 640625 630 635 640
Gln Gly Leu His Lys Gly Gln Ser Ser His Ser Ala Gly Pro Asn GlyGln Gly Leu His Lys Gly Gln Ser Ser His Ser Ala Gly Pro Asn Gly
645 650 655 645 650 655
Glu Arg Pro Leu Ser Ser Thr Gly Pro Ser Gln His Leu Gln Ala AlaGlu Arg Pro Leu Ser Ser Thr Gly Pro Ser Gln His Leu Gln Ala Ala
660 665 670 660 665 670
Gly Ser Gly Ile Gln Asn Gln Asn Gly His Pro Thr Leu Pro Ser AsnGly Ser Gly Ile Gln Asn Gln Asn Gly His Pro Thr Leu Pro Ser Asn
675 680 685 675 680 685
Ser Val Thr Gln Gly Ala Ala Leu Asn His Leu Ser Ser His Thr AlaSer Val Thr Gln Gly Ala Ala Leu Asn His Leu Ser Ser His Thr Ala
690 695 700 690 695 700
Thr Ser Gly Gly Gln Gln Gly Ile Thr Leu Thr Lys Glu Ser Lys ProThr Ser Gly Gly Gln Gln Gly Ile Thr Leu Thr Lys Glu Ser Lys Pro
705 710 715 720705 710 715 720
Ser Gly Asn Ile Leu Thr Val Pro Glu Thr Ser Arg His Thr Gly GluSer Gly Asn Ile Leu Thr Val Pro Glu Thr Ser Arg His Thr Gly Glu
725 730 735 725 730 735
Thr Pro Asn Ser Thr Ala Ser Val Glu Gly Leu Pro Asn His Val HisThr Pro Asn Ser Thr Ala Ser Val Glu Gly Leu Pro Asn His Val His
740 745 750 740 745 750
Gln Met Thr Ala Asp Ala Val Cys Ser Pro Ser His Gly Asp Ser LysGln Met Thr Ala Asp Ala Val Cys Ser Pro Ser His Gly Asp Ser Lys
755 760 765 755 760 765
Ser Pro Gly Leu Leu Ser Ser Asp Asn Pro Gln Leu Ser Ala Leu LeuSer Pro Gly Leu Leu Ser Ser Asp Asn Pro Gln Leu Ser Ala Leu Leu
770 775 780 770 775 780
Met Gly Lys Ala Asn Asn Asn Val Gly Thr Gly Thr Cys Asp Lys ValMet Gly Lys Ala Asn Asn Asn Val Gly Thr Gly Thr Cys Asp Lys Val
785 790 795 800785 790 795 800
Asn Asn Ile His Pro Ala Val His Thr Lys Thr Asp Asn Ser Val AlaAsn Asn Ile His Pro Ala Val His Thr Lys Thr Asp Asn Ser Val Ala
805 810 815 805 810 815
Ser Ser Pro Ser Ser Ala Ile Ser Thr Ala Thr Pro Ser Pro Lys SerSer Ser Pro Ser Ser Ala Ile Ser Thr Ala Thr Pro Ser Pro Lys Ser
820 825 830 820 825 830
Thr Glu Gln Thr Thr Thr Asn Ser Val Thr Ser Leu Asn Ser Pro HisThr Glu Gln Thr Thr Thr Asn Ser Val Thr Ser Leu Asn Ser Pro His
835 840 845 835 840 845
Ser Gly Leu His Thr Ile Asn Gly Glu Gly Met Glu Glu Ser Gln SerSer Gly Leu His Thr Ile Asn Gly Glu Gly Met Glu Glu Ser Gln Ser
850 855 860 850 855 860
Pro Met Lys Thr Asp Leu Leu Leu Val Asn His Lys Pro Ser Pro GlnPro Met Lys Thr Asp Leu Leu Leu Val Asn His Lys Pro Ser Pro Gln
865 870 875 880865 870 875 880
Ile Ile Pro Ser Met Ser Val Ser Ile Tyr Pro Ser Ser Ala Glu ValIle Ile Pro Ser Met Ser Val Ser Ile Tyr Pro Ser Ser Ala Glu Val
885 890 895 885 890 895
Leu Lys Ala Cys Arg Asn Leu Gly Lys Asn Gly Leu Ser Asn Ser SerLeu Lys Ala Cys Arg Asn Leu Gly Lys Asn Gly Leu Ser Asn Ser Ser
900 905 910 900 905 910
Ile Leu Leu Asp Lys Cys Pro Pro Pro Arg Pro Pro Ser Ser Pro TyrIle Leu Leu Asp Lys Cys Pro Pro Pro Arg Pro Pro Ser Ser Pro Tyr
915 920 925 915 920 925
Pro Pro Leu Pro Lys Asp Lys Leu Asn Pro Pro Thr Pro Ser Ile TyrPro Pro Leu Pro Lys Asp Lys Leu Asn Pro Pro Thr Pro Ser Ile Tyr
930 935 940 930 935 940
Leu Glu Asn Lys Arg Asp Ala Phe Phe Pro Pro Leu His Gln Phe CysLeu Glu Asn Lys Arg Asp Ala Phe Phe Pro Pro Leu His Gln Phe Cys
945 950 955 960945 950 955 960
Thr Asn Pro Asn Asn Pro Val Thr Val Ile Arg Gly Leu Ala Gly AlaThr Asn Pro Asn Asn Pro Val Thr Val Ile Arg Gly Leu Ala Gly Ala
965 970 975 965 970 975
Leu Lys Leu Asp Leu Gly Leu Phe Ser Thr Lys Thr Leu Val Glu AlaLeu Lys Leu Asp Leu Gly Leu Phe Ser Thr Lys Thr Leu Val Glu Ala
980 985 990 980 985 990
Asn Asn Glu His Met Val Glu Val Arg Thr Gln Leu Leu Gln Pro AlaAsn Asn Glu His Met Val Glu Val Arg Thr Gln Leu Leu Gln Pro Ala
995 1000 1005 995 1000 1005
Asp Glu Asn Trp Asp Pro Thr Gly Thr Lys Lys Ile Trp His CysAsp Glu Asn Trp Asp Pro Thr Gly Thr Lys Lys Ile Trp His Cys
1010 1015 1020 1010 1015 1020
Glu Ser Asn Arg Ser His Thr Thr Ile Ala Lys Tyr Ala Gln TyrGlu Ser Asn Arg Ser His Thr Thr Ile Ala Lys Tyr Ala Gln Tyr
1025 1030 1035 1025 1030 1035
Gln Ala Ser Ser Phe Gln Glu Ser Leu Arg Glu Glu Asn Glu LysGln Ala Ser Ser Phe Gln Glu Ser Leu Arg Glu Glu Asn Glu Lys
1040 1045 1050 1040 1045 1050
Arg Ser His His Lys Asp His Ser Asp Ser Glu Ser Thr Ser SerArg Ser His His Lys Asp His Ser Asp Ser Glu Ser Thr Ser Ser
1055 1060 1065 1055 1060 1065
Asp Asn Ser Gly Arg Arg Arg Lys Gly Pro Phe Lys Thr Ile LysAsp Asn Ser Gly Arg Arg Arg Lys Gly Pro Phe Lys Thr Ile Lys
1070 1075 1080 1070 1075 1080
Phe Gly Thr Asn Ile Asp Leu Ser Asp Asp Lys Lys Trp Lys LeuPhe Gly Thr Asn Ile Asp Leu Ser Asp Asp Lys Lys Trp Lys Leu
1085 1090 1095 1085 1090 1095
Gln Leu His Glu Leu Thr Lys Leu Pro Ala Phe Val Arg Val ValGln Leu His Glu Leu Thr Lys Leu Pro Ala Phe Val Arg Val Val
1100 1105 1110 1100 1105 1110
Ser Ala Gly Asn Leu Leu Ser His Val Gly His Thr Ile Leu GlySer Ala Gly Asn Leu Leu Ser His Val Gly His Thr Ile Leu Gly
1115 1120 1125 1115 1120 1125
Met Asn Thr Val Gln Leu Tyr Met Lys Val Pro Gly Ser Arg ThrMet Asn Thr Val Gln Leu Tyr Met Lys Val Pro Gly Ser Arg Thr
1130 1135 1140 1130 1135 1140
Pro Gly His Gln Glu Asn Asn Asn Phe Cys Ser Val Asn Ile AsnPro Gly His Gln Glu Asn Asn Asn Phe Cys Ser Val Asn Ile Asn
1145 1150 1155 1145 1150 1155
Ile Gly Pro Gly Asp Cys Glu Trp Phe Val Val Pro Glu Gly TyrIle Gly Pro Gly Asp Cys Glu Trp Phe Val Val Pro Glu Gly Tyr
1160 1165 1170 1160 1165 1170
Trp Gly Val Leu Asn Asp Phe Cys Glu Lys Asn Asn Leu Asn PheTrp Gly Val Leu Asn Asp Phe Cys Glu Lys Asn Asn Leu Asn Phe
1175 1180 1185 1175 1180 1185
Leu Met Gly Ser Trp Trp Pro Asn Leu Glu Asp Leu Tyr Glu AlaLeu Met Gly Ser Trp Trp Pro Asn Leu Glu Asp Leu Tyr Glu Ala
1190 1195 1200 1190 1195 1200
Asn Val Pro Val Tyr Arg Phe Ile Gln Arg Pro Gly Asp Leu ValAsn Val Pro Val Tyr Arg Phe Ile Gln Arg Pro Gly Asp Leu Val
1205 1210 1215 1205 1210 1215
Trp Ile Asn Ala Gly Thr Val His Trp Val Gln Ala Ile Gly TrpTrp Ile Asn Ala Gly Thr Val His Trp Val Gln Ala Ile Gly Trp
1220 1225 1230 1220 1225 1230
Cys Asn Asn Ile Ala Trp Asn Val Gly Pro Leu Thr Ala Cys GlnCys Asn Asn Ile Ala Trp Asn Val Gly Pro Leu Thr Ala Cys Gln
1235 1240 1245 1235 1240 1245
Tyr Lys Leu Ala Val Glu Arg Tyr Glu Trp Asn Lys Leu Gln SerTyr Lys Leu Ala Val Glu Arg Tyr Glu Trp Asn Lys Leu Gln Ser
1250 1255 1260 1250 1255 1260
Val Lys Ser Ile Val Pro Met Val His Leu Ser Trp Asn Met AlaVal Lys Ser Ile Val Pro Met Val His Leu Ser Trp Asn Met Ala
1265 1270 1275 1265 1270 1275
Arg Asn Ile Lys Val Ser Asp Pro Lys Leu Phe Glu Met Ile LysArg Asn Ile Lys Val Ser Asp Pro Lys Leu Phe Glu Met Ile Lys
1280 1285 1290 1280 1285 1290
Tyr Cys Leu Leu Arg Thr Leu Lys Gln Cys Gln Thr Leu Arg GluTyr Cys Leu Leu Arg Thr Leu Lys Gln Cys Gln Thr Leu Arg Glu
1295 1300 1305 1295 1300 1305
Ala Leu Ile Ala Ala Gly Lys Glu Ile Ile Trp His Gly Arg ThrAla Leu Ile Ala Ala Gly Lys Glu Ile Ile Trp His Gly Arg Thr
1310 1315 1320 1310 1315 1320
Lys Glu Glu Pro Ala His Tyr Cys Ser Ile Cys Glu Val Glu ValLys Glu Glu Pro Ala His Tyr Cys Ser Ile Cys Glu Val Glu Val
1325 1330 1335 1325 1330 1335
Phe Asp Leu Leu Phe Val Thr Asn Glu Ser Asn Ser Arg Lys ThrPhe Asp Leu Leu Phe Val Thr Asn Glu Ser Asn Ser Arg Lys Thr
1340 1345 1350 1340 1345 1350
Tyr Ile Val His Cys Gln Asp Cys Ala Arg Lys Thr Ser Gly AsnTyr Ile Val His Cys Gln Asp Cys Ala Arg Lys Thr Ser Gly Asn
1355 1360 1365 1355 1360 1365
Leu Glu Asn Phe Val Val Leu Glu Gln Tyr Lys Met Glu Asp LeuLeu Glu Asn Phe Val Val Leu Glu Gln Tyr Lys Met Glu Asp Leu
1370 1375 1380 1370 1375 1380
Met Gln Val Tyr Asp Gln Phe Thr Leu Ala Pro Pro Leu Pro SerMet Gln Val Tyr Asp Gln Phe Thr Leu Ala Pro Pro Leu Pro Ser
1385 1390 1395 1385 1390 1395
Ala Ser SerAla Ser Ser
1400 1400
<210> 3<210> 3
<211> 5918<211> 5918
<212> DNA<212>DNA
<213> 小鼠(Mus musculus)<213> Mouse (Mus musculus)
<220><220>
<221> CDS<221> CDS
<222> (432)..(4637)<222> (432)..(4637)
<400> 3<400> 3
ggctgcgccg agcgggctgt gagggggggg tcacggcagc ggcgtggggt tcgctgtgtg 60ggctgcgccg agcgggctgt gagggggggg tcacggcagc ggcgtggggt tcgctgtgtg 60
acacaattac aacaactttg tgctggtgcc ggggaagttt gtgtctccta cgaatccccc 120acacaattac aacaactttg tgctggtgcc ggggaagttt gtgtctccta cgaatccccc 120
ccagagctcc cgctcctcgc gcgctccggc cattcccgcc gaccctgcct gtggctgccc 180ccagagctcc cgctcctcgc gcgctccggc cattcccgcc gaccctgcct gtggctgccc 180
ccttcccatc cccgcgatgt gaccctacag ccgagccgtc gccgctccgg acccagtggt 240ccttcccatc cccgcgatgt gaccctacag ccgagccgtc gccgctccgg accccagtggt 240
tccacagcct ctgctgcctc tgccttcacc tctaccgcgt taggattttt cgctgccgcc 300tccacagcct ctgctgcctc tgccttcacc tctaccgcgt taggattttt cgctgccgcc 300
tctcgcggca gaggaggagg cagaaataaa gttggtgtgc cggtcttgtg cggagattgg 360tctcgcggca gaggaggagg cagaaataaa gttggtgtgc cggtcttgtg cggagattgg 360
aggcgtcctt gcgggctccg gcccggagag ggggggtgtc ggcgttggag ttgtgaattc 420aggcgtcctt gcgggctccg gcccggagag ggggggtgtc ggcgttggag ttgtgaattc 420
gctgcgtttc c atg aaa tcc tgc gga gtg tcg ctc gct acc gcc gcc gcc 470gctgcgtttc c atg aaa tcc tgc gga gtg tcg ctc gct acc gcc gcc gcc 470
Met Lys Ser Cys Gly Val Ser Leu Ala Thr Ala Ala Ala Met Lys Ser Cys Gly Val Ser Leu Ala Thr Ala Ala Ala
1 5 10 1 5 10
gcc gcc gcc gcc gcc gct ttc ggt gat gag gaa aag aaa atg gcg gcg 518gcc gcc gcc gcc gcc gct ttc ggt gat gag gaa aag aaa atg gcg gcg 518
Ala Ala Ala Ala Ala Ala Phe Gly Asp Glu Glu Lys Lys Met Ala AlaAla Ala Ala Ala Ala Ala Phe Gly Asp Glu Glu Lys Lys Met Ala Ala
15 20 25 15 20 25
gga aaa gcg agc ggc gag agc gag gag gcg tcc ccc agc ctg aca gcg 566gga aaa gcg agc ggc gag agc gag gag gcg tcc ccc agc ctg aca gcg 566
Gly Lys Ala Ser Gly Glu Ser Glu Glu Ala Ser Pro Ser Leu Thr AlaGly Lys Ala Ser Gly Glu Ser Glu Glu Ala Ser Pro Ser Leu Thr Ala
30 35 40 4530 35 40 45
gag gag agg gag gcg ctc ggc gga ctg gac agc cgc ctt ttc ggg ttc 614gag gag agg gag gcg ctc ggc gga ctg gac agc cgc ctt ttc ggg ttc 614
Glu Glu Arg Glu Ala Leu Gly Gly Leu Asp Ser Arg Leu Phe Gly PheGlu Glu Arg Glu Ala Leu Gly Gly Leu Asp Ser Arg Leu Phe Gly Phe
50 55 60 50 55 60
gtg agg ttt cat gaa gat ggc gcc agg atg aag gcc ctg ctg ggc aag 662gtg agg ttt cat gaa gat ggc gcc agg atg aag gcc ctg ctg ggc aag 662
Val Arg Phe His Glu Asp Gly Ala Arg Met Lys Ala Leu Leu Gly LysVal Arg Phe His Glu Asp Gly Ala Arg Met Lys Ala Leu Leu Gly Lys
65 70 75 65 70 75
gct gtt cgc tgc tac gaa tct cta atc tta aaa gct gaa ggg aaa gtg 710gct gtt cgc tgc tac gaa tct cta atc tta aaa gct gaa ggg aaa gtg 710
Ala Val Arg Cys Tyr Glu Ser Leu Ile Leu Lys Ala Glu Gly Lys ValAla Val Arg Cys Tyr Glu Ser Leu Ile Leu Lys Ala Glu Gly Lys Val
80 85 90 80 85 90
gag tct gat ttc ttt tgt caa tta ggt cac ttc aac ctc tta ttg gaa 758gag tct gat ttc ttt tgt caa tta ggt cac ttc aac ctc tta ttg gaa 758
Glu Ser Asp Phe Phe Cys Gln Leu Gly His Phe Asn Leu Leu Leu GluGlu Ser Asp Phe Phe Cys Gln Leu Gly His Phe Asn Leu Leu Leu Glu
95 100 105 95 100 105
gat tat cca aaa gca tta tct gca tac cag agg tac tac agt tta cag 806gat tat cca aaa gca tta tct gca tac cag agg tac tac agt tta cag 806
Asp Tyr Pro Lys Ala Leu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu GlnAsp Tyr Pro Lys Ala Leu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu Gln
110 115 120 125110 115 120 125
tct gat tac tgg aag aat gct gcc ttt tta tat ggt ctt ggt ttg gtc 854tct gat tac tgg aag aat gct gcc ttt tta tat ggt ctt ggt ttg gtc 854
Ser Asp Tyr Trp Lys Asn Ala Ala Phe Leu Tyr Gly Leu Gly Leu ValSer Asp Tyr Trp Lys Asn Ala Ala Phe Leu Tyr Gly Leu Gly Leu Val
130 135 140 130 135 140
tac ttc cat tac aat gca ttt cag tgg gct att aaa gca ttt cag gag 902tac ttc cat tac aat gca ttt cag tgg gct att aaa gca ttt cag gag 902
Tyr Phe His Tyr Asn Ala Phe Gln Trp Ala Ile Lys Ala Phe Gln GluTyr Phe His Tyr Asn Ala Phe Gln Trp Ala Ile Lys Ala Phe Gln Glu
145 150 155 145 150 155
gtg ctt tat gtc gat ccc agc ttt tgt cga gcc aag gaa att cat tta 950gtg ctt tat gtc gat ccc agc ttt tgt cga gcc aag gaa att cat tta 950
Val Leu Tyr Val Asp Pro Ser Phe Cys Arg Ala Lys Glu Ile His LeuVal Leu Tyr Val Asp Pro Ser Phe Cys Arg Ala Lys Glu Ile His Leu
160 165 170 160 165 170
cga ctt ggg ctt atg ttc aaa gtg aac aca gac tat gag tct agt tta 998cga ctt ggg ctt atg ttc aaa gtg aac aca gac tat gag tct agt tta 998
Arg Leu Gly Leu Met Phe Lys Val Asn Thr Asp Tyr Glu Ser Ser LeuArg Leu Gly Leu Met Phe Lys Val Asn Thr Asp Tyr Glu Ser Ser Leu
175 180 185 175 180 185
aag cat ttt cag tta gct ttg gtt gac tgt aat ccc tgc act ttg tcc 1046aag cat ttt cag tta gct ttg gtt gac tgt aat ccc tgc act ttg tcc 1046
Lys His Phe Gln Leu Ala Leu Val Asp Cys Asn Pro Cys Thr Leu SerLys His Phe Gln Leu Ala Leu Val Asp Cys Asn Pro Cys Thr Leu Ser
190 195 200 205190 195 200 205
aat gct gaa att cag ttt cac att gcc cac tta tat gaa acc cag agg 1094aat gct gaa att cag ttt cac att gcc cac tta tat gaa acc cag agg 1094
Asn Ala Glu Ile Gln Phe His Ile Ala His Leu Tyr Glu Thr Gln ArgAsn Ala Glu Ile Gln Phe His Ile Ala His Leu Tyr Glu Thr Gln Arg
210 215 220 210 215 220
aag tat cat tct gca aaa gaa gct tat gag caa ctt ttg cag aca gaa 1142aag tat cat tct gca aaa gaa gct tat gag caa ctt ttg cag aca gaa 1142
Lys Tyr His Ser Ala Lys Glu Ala Tyr Glu Gln Leu Leu Gln Thr GluLys Tyr His Ser Ala Lys Glu Ala Tyr Glu Gln Leu Leu Gln Thr Glu
225 230 235 225 230 235
aac ctt tct gca caa gta aaa gca act att tta caa caa tta ggt tgg 1190aac ctt tct gca caa gta aaa gca act att tta caa caa tta ggt tgg 1190
Asn Leu Ser Ala Gln Val Lys Ala Thr Ile Leu Gln Gln Leu Gly TrpAsn Leu Ser Ala Gln Val Lys Ala Thr Ile Leu Gln Gln Leu Gly Trp
240 245 250 240 245 250
atg cat cac act gtg gat ctc ctg gga gat aag gcc acc aag gaa agt 1238atg cat cac act gtg gat ctc ctg gga gat aag gcc acc aag gaa agt 1238
Met His His Thr Val Asp Leu Leu Gly Asp Lys Ala Thr Lys Glu SerMet His His Thr Val Asp Leu Leu Gly Asp Lys Ala Thr Lys Glu Ser
255 260 265 255 260 265
tat gct att cag tat ctc cag aag tcc ttg gaa gca gat cca aat tct 1286tat gct att cag tat ctc cag aag tcc ttg gaa gca gat cca aat tct 1286
Tyr Ala Ile Gln Tyr Leu Gln Lys Ser Leu Glu Ala Asp Pro Asn SerTyr Ala Ile Gln Tyr Leu Gln Lys Ser Leu Glu Ala Asp Pro Asn Ser
270 275 280 285270 275 280 285
ggc cag tcc tgg tat ttc ctt gga agg tgc tat tca agt att ggg aaa 1334ggc cag tcc tgg tat ttc ctt gga agg tgc tat tca agt att ggg aaa 1334
Gly Gln Ser Trp Tyr Phe Leu Gly Arg Cys Tyr Ser Ser Ile Gly LysGly Gln Ser Trp Tyr Phe Leu Gly Arg Cys Tyr Ser Ser Ile Gly Lys
290 295 300 290 295 300
gtt cag gat gcc ttt ata tct tac agg caa tct att gat aaa tca gaa 1382gtt cag gat gcc ttt ata tct tac agg caa tct att gat aaa tca gaa 1382
Val Gln Asp Ala Phe Ile Ser Tyr Arg Gln Ser Ile Asp Lys Ser GluVal Gln Asp Ala Phe Ile Ser Tyr Arg Gln Ser Ile Asp Lys Ser Glu
305 310 315 305 310 315
gca agt gca gat aca tgg tgt tca ata ggt gtg ctc tat caa cag caa 1430gca agt gca gat aca tgg tgt tca ata ggt gtg ctc tat caa cag caa 1430
Ala Ser Ala Asp Thr Trp Cys Ser Ile Gly Val Leu Tyr Gln Gln GlnAla Ser Ala Asp Thr Trp Cys Ser Ile Gly Val Leu Tyr Gln Gln Gln
320 325 330 320 325 330
aat cag cct atg gat gct ttg caa gct tat att tgt gct gta caa ttg 1478aat cag cct atg gat gct ttg caa gct tat att tgt gct gta caa ttg 1478
Asn Gln Pro Met Asp Ala Leu Gln Ala Tyr Ile Cys Ala Val Gln LeuAsn Gln Pro Met Asp Ala Leu Gln Ala Tyr Ile Cys Ala Val Gln Leu
335 340 345 335 340 345
gac cac ggt cat gct gca gcc tgg atg gat cta ggc act ctc tat gaa 1526gac cac ggt cat gct gca gcc tgg atg gat cta ggc act ctc tat gaa 1526
Asp His Gly His Ala Ala Ala Trp Met Asp Leu Gly Thr Leu Tyr GluAsp His Gly His Ala Ala Ala Trp Met Asp Leu Gly Thr Leu Tyr Glu
350 355 360 365350 355 360 365
tcc tgc aac caa cct cag gat gct att aaa tgc tat tta aat gca act 1574tcc tgc aac caa cct cag gat gct att aaa tgc tat tta aat gca act 1574
Ser Cys Asn Gln Pro Gln Asp Ala Ile Lys Cys Tyr Leu Asn Ala ThrSer Cys Asn Gln Pro Gln Asp Ala Ile Lys Cys Tyr Leu Asn Ala Thr
370 375 380 370 375 380
aga agc aaa aat tgt agt aat acc tct gga ctt gca gca cga att aag 1622aga agc aaa aat tgt agt aat acc tct gga ctt gca gca cga att aag 1622
Arg Ser Lys Asn Cys Ser Asn Thr Ser Gly Leu Ala Ala Arg Ile LysArg Ser Lys Asn Cys Ser Asn Thr Ser Gly Leu Ala Ala Arg Ile Lys
385 390 395 385 390 395
tat tta cag gct cag ttg tgt aac ctt cca caa ggt agt cta cag aat 1670tat tta cag gct cag ttg tgt aac ctt cca caa ggt agt cta cag aat 1670
Tyr Leu Gln Ala Gln Leu Cys Asn Leu Pro Gln Gly Ser Leu Gln AsnTyr Leu Gln Ala Gln Leu Cys Asn Leu Pro Gln Gly Ser Leu Gln Asn
400 405 410 400 405 410
aaa act aaa tta ctt cct agt att gag gag gca tgg agc cta cca atc 1718aaa act aaa tta ctt cct agt att gag gag gca tgg agc cta cca atc 1718
Lys Thr Lys Leu Leu Pro Ser Ile Glu Glu Ala Trp Ser Leu Pro IleLys Thr Lys Leu Leu Pro Ser Ile Glu Glu Ala Trp Ser Leu Pro Ile
415 420 425 415 420 425
ccc gca gag ctt acc tcc agg cag ggt gcc atg aac aca gca cag cag 1766ccc gca gag ctt acc tcc agg cag ggt gcc atg aac aca gca cag cag 1766
Pro Ala Glu Leu Thr Ser Arg Gln Gly Ala Met Asn Thr Ala Gln GlnPro Ala Glu Leu Thr Ser Arg Gln Gly Ala Met Asn Thr Ala Gln Gln
430 435 440 445430 435 440 445
aat act tct gat aat tgg agt ggt ggc aat gca cca cct cca gta gaa 1814aat act tct gat aat tgg agt ggt ggc aat gca cca cct cca gta gaa 1814
Asn Thr Ser Asp Asn Trp Ser Gly Gly Asn Ala Pro Pro Pro Val GluAsn Thr Ser Asp Asn Trp Ser Gly Gly Asn Ala Pro Pro Pro Pro Val Glu
450 455 460 450 455 460
caa caa act cat tca tgg tgt ttg aca cca cag aaa tta cag cac ttg 1862caa caa act cat tca tgg tgt ttg aca cca cag aaa tta cag cac ttg 1862
Gln Gln Thr His Ser Trp Cys Leu Thr Pro Gln Lys Leu Gln His LeuGln Gln Thr His Ser Trp Cys Leu Thr Pro Gln Lys Leu Gln His Leu
465 470 475 465 470 475
gaa cag ctc cga gca aac aga aat aat tta aat cca gca cag aaa cta 1910gaa cag ctc cga gca aac aga aat aat tta aat cca gca cag aaa cta 1910
Glu Gln Leu Arg Ala Asn Arg Asn Asn Leu Asn Pro Ala Gln Lys LeuGlu Gln Leu Arg Ala Asn Arg Asn Asn Leu Asn Pro Ala Gln Lys Leu
480 485 490 480 485 490
atg ctg gaa cag ctg gaa agt cag ttt gtc tta atg cag caa cac caa 1958atg ctg gaa cag ctg gaa agt cag ttt gtc tta atg cag caa cac caa 1958
Met Leu Glu Gln Leu Glu Ser Gln Phe Val Leu Met Gln Gln His GlnMet Leu Glu Gln Leu Glu Ser Gln Phe Val Leu Met Gln Gln His Gln
495 500 505 495 500 505
atg aga caa aca gga gtt gca cag gta cgg cct act gga att ctt aat 2006atg aga caa aca gga gtt gca cag gta cgg cct act gga att ctt aat 2006
Met Arg Gln Thr Gly Val Ala Gln Val Arg Pro Thr Gly Ile Leu AsnMet Arg Gln Thr Gly Val Ala Gln Val Arg Pro Thr Gly Ile Leu Asn
510 515 520 525510 515 520 525
ggg cca aca gtt gac tca tca ctg cct aca aac tca gtt tct ggc cag 2054ggg cca aca gtt gac tca tca ctg cct aca aac tca gtt tct ggc cag 2054
Gly Pro Thr Val Asp Ser Ser Leu Pro Thr Asn Ser Val Ser Gly GlnGly Pro Thr Val Asp Ser Ser Leu Pro Thr Asn Ser Val Ser Gly Gln
530 535 540 530 535 540
cag cca cag ctt cct ctg acc aga atg cct agt gtc tct cag cct gga 2102cag cca cag ctt cct ctg acc aga atg cct agt gtc tct cag cct gga 2102
Gln Pro Gln Leu Pro Leu Thr Arg Met Pro Ser Val Ser Gln Pro GlyGln Pro Gln Leu Pro Leu Thr Arg Met Pro Ser Val Ser Gln Pro Gly
545 550 555 545 550 555
gtc cac act gcc tgt cct agg cag act ttg gcc aat gga ccc ttt tct 2150gtc cac act gcc tgt cct agg cag act ttg gcc aat gga ccc ttt tct 2150
Val His Thr Ala Cys Pro Arg Gln Thr Leu Ala Asn Gly Pro Phe SerVal His Thr Ala Cys Pro Arg Gln Thr Leu Ala Asn Gly Pro Phe Ser
560 565 570 560 565 570
gca ggc cat gtt ccc tgt agc aca tca aga aca ctg gga agt aca gac 2198gca ggc cat gtt ccc tgt agc aca tca aga aca ctg gga agt aca gac 2198
Ala Gly His Val Pro Cys Ser Thr Ser Arg Thr Leu Gly Ser Thr AspAla Gly His Val Pro Cys Ser Thr Ser Arg Thr Leu Gly Ser Thr Asp
575 580 585 575 580 585
act gtt ttg ata ggc aat aat cat gta aca gga agt gga agt aat gga 2246act gtt ttg ata ggc aat aat cat gta aca gga agt gga agt aat gga 2246
Thr Val Leu Ile Gly Asn Asn His Val Thr Gly Ser Gly Ser Asn GlyThr Val Leu Ile Gly Asn Asn His Val Thr Gly Ser Gly Ser Asn Gly
590 595 600 605590 595 600 605
aac gtg cct tac ctg cag cga aac gca ccc act cta cct cat aac cgc 2294aac gtg cct tac ctg cag cga aac gca ccc act cta cct cat aac cgc 2294
Asn Val Pro Tyr Leu Gln Arg Asn Ala Pro Thr Leu Pro His Asn ArgAsn Val Pro Tyr Leu Gln Arg Asn Ala Pro Thr Leu Pro His Asn Arg
610 615 620 610 615 620
aca aac ctg acc agc agc aca gag gag ccg tgg aaa aac caa cta tct 2342aca aac ctg acc agc agc aca gag gag ccg tgg aaa aac caa cta tct 2342
Thr Asn Leu Thr Ser Ser Thr Glu Glu Pro Trp Lys Asn Gln Leu SerThr Asn Leu Thr Ser Ser Thr Glu Glu Pro Trp Lys Asn Gln Leu Ser
625 630 635 625 630 635
aac tcc act cag ggg ctt cac aaa ggt ccg agt tca cat ttg gca ggt 2390aac tcc act cag ggg ctt cac aaa ggt ccg agt tca cat ttg gca ggt 2390
Asn Ser Thr Gln Gly Leu His Lys Gly Pro Ser Ser His Leu Ala GlyAsn Ser Thr Gln Gly Leu His Lys Gly Pro Ser Ser His Leu Ala Gly
640 645 650 640 645 650
cct aat ggt gaa cga cct cta tct tcc act ggg cct tcc cag cat ctc 2438cct aat ggt gaa cga cct cta tct tcc act ggg cct tcc cag cat ctc 2438
Pro Asn Gly Glu Arg Pro Leu Ser Ser Thr Gly Pro Ser Gln His LeuPro Asn Gly Glu Arg Pro Leu Ser Ser Thr Gly Pro Ser Gln His Leu
655 660 665 655 660 665
cag gca gct ggc tct ggt att cag aat cag aat gga cat ccc acc ctg 2486cag gca gct ggc tct ggt att cag aat cag aat gga cat ccc acc ctg 2486
Gln Ala Ala Gly Ser Gly Ile Gln Asn Gln Asn Gly His Pro Thr LeuGln Ala Ala Gly Ser Gly Ile Gln Asn Gln Asn Gly His Pro Thr Leu
670 675 680 685670 675 680 685
cct agc aat tca gta aca cag ggg gct gct ctc aat cac ctc tcc tct 2534cct agc aat tca gta aca cag ggg gct gct ctc aat cac ctc tcc tct 2534
Pro Ser Asn Ser Val Thr Gln Gly Ala Ala Leu Asn His Leu Ser SerPro Ser Asn Ser Val Thr Gln Gly Ala Ala Leu Asn His Leu Ser Ser
690 695 700 690 695 700
cac act gct acc tca ggt gga caa caa ggc att acc tta acc aaa gag 2582cac act gct acc tca ggt gga caa caa ggc att acc tta acc aaa gag 2582
His Thr Ala Thr Ser Gly Gly Gln Gln Gly Ile Thr Leu Thr Lys GluHis Thr Ala Thr Ser Gly Gly Gln Gln Gly Ile Thr Leu Thr Lys Glu
705 710 715 705 710 715
agc aag cct tca gga aac aca ttg acg gtg cct gaa aca agc agg caa 2630agc aag cct tca gga aac aca ttg acg gtg cct gaa aca agc agg caa 2630
Ser Lys Pro Ser Gly Asn Thr Leu Thr Val Pro Glu Thr Ser Arg GlnSer Lys Pro Ser Gly Asn Thr Leu Thr Val Pro Glu Thr Ser Arg Gln
720 725 730 720 725 730
act gga gag aca cct aac agc act gcc agt gtt gag gga ctt cct aat 2678act gga gag aca cct aac agc act gcc agt gtt gag gga ctt cct aat 2678
Thr Gly Glu Thr Pro Asn Ser Thr Ala Ser Val Glu Gly Leu Pro AsnThr Gly Glu Thr Pro Asn Ser Thr Ala Ser Val Glu Gly Leu Pro Asn
735 740 745 735 740 745
cat gtc cat cag gtg atg gca gat gct gtt tgc agt cct agc cat gga 2726cat gtc cat cag gtg atg gca gat gct gtt tgc agt cct agc cat gga 2726
His Val His Gln Val Met Ala Asp Ala Val Cys Ser Pro Ser His GlyHis Val His Gln Val Met Ala Asp Ala Val Cys Ser Pro Ser His Gly
750 755 760 765750 755 760 765
gat tct aag tca cca ggt tta cta agt tca gac aat cct cag ctc tct 2774gat tct aag tca cca ggt tta cta agt tca gac aat cct cag ctc tct 2774
Asp Ser Lys Ser Pro Gly Leu Leu Ser Ser Asp Asn Pro Gln Leu SerAsp Ser Lys Ser Pro Gly Leu Leu Ser Ser Asp Asn Pro Gln Leu Ser
770 775 780 770 775 780
gcc ttg ttg atg gga aaa gct aat aac aat gtg ggt cct gga acc tgt 2822gcc ttg ttg atg gga aaa gct aat aac aat gtg ggt cct gga acc tgt 2822
Ala Leu Leu Met Gly Lys Ala Asn Asn Asn Val Gly Pro Gly Thr CysAla Leu Leu Met Gly Lys Ala Asn Asn Asn Val Gly Pro Gly Thr Cys
785 790 795 785 790 795
gac aaa gtc aat aac atc cac cca act gtc cat aca aag act gat aat 2870gac aaa gtc aat aac atc cac cca act gtc cat aca aag act gat aat 2870
Asp Lys Val Asn Asn Ile His Pro Thr Val His Thr Lys Thr Asp AsnAsp Lys Val Asn Asn Ile His Pro Thr Val His Thr Lys Thr Asp Asn
800 805 810 800 805 810
tct gtt gcc tct tca cca tct tca gcc att tcc aca gca aca cct tct 2918tct gtt gcc tct tca cca tct tca gcc att tcc aca gca aca cct tct 2918
Ser Val Ala Ser Ser Pro Ser Ser Ala Ile Ser Thr Ala Thr Pro SerSer Val Ala Ser Ser Pro Ser Ser Ala Ile Ser Thr Ala Thr Pro Ser
815 820 825 815 820 825
cct aag tcc act gaa cag aca acc aca aac agt gtt acc agc ctt aac 2966cct aag tcc act gaa cag aca acc aca aac agt gtt acc agc ctt aac 2966
Pro Lys Ser Thr Glu Gln Thr Thr Thr Asn Ser Val Thr Ser Leu AsnPro Lys Ser Thr Glu Gln Thr Thr Thr Asn Ser Val Thr Ser Leu Asn
830 835 840 845830 835 840 845
agc cct cac agt ggg ctg cac aca att aat gga gaa gga atg gaa gaa 3014agc cct cac agt ggg ctg cac aca att aat gga gaa gga atg gaa gaa 3014
Ser Pro His Ser Gly Leu His Thr Ile Asn Gly Glu Gly Met Glu GluSer Pro His Ser Gly Leu His Thr Ile Asn Gly Glu Gly Met Glu Glu
850 855 860 850 855 860
tct cag agc ccc att aaa aca gat ctg ctt cta gtt agc cac aga cct 3062tct cag agc ccc att aaa aca gat ctg ctt cta gtt agc cac aga cct 3062
Ser Gln Ser Pro Ile Lys Thr Asp Leu Leu Leu Val Ser His Arg ProSer Gln Ser Pro Ile Lys Thr Asp Leu Leu Leu Val Ser His Arg Pro
865 870 875 865 870 875
agt cct cag atc ata cca tca atg tct gtg tcc ata tat ccc agc tca 3110agt cct cag atc ata cca tca atg tct gtg tcc ata tat ccc agc tca 3110
Ser Pro Gln Ile Ile Pro Ser Met Ser Val Ser Ile Tyr Pro Ser SerSer Pro Gln Ile Ile Pro Ser Met Ser Val Ser Ile Tyr Pro Ser Ser
880 885 890 880 885 890
gca gaa gtt ctg aaa gct tgc agg aat cta ggt aaa aac ggc ctg tct 3158gca gaa gtt ctg aaa gct tgc agg aat cta ggt aaa aac ggc ctg tct 3158
Ala Glu Val Leu Lys Ala Cys Arg Asn Leu Gly Lys Asn Gly Leu SerAla Glu Val Leu Lys Ala Cys Arg Asn Leu Gly Lys Asn Gly Leu Ser
895 900 905 895 900 905
aat agt agc att ctg ttg gat aaa tgt ccg cct cca aga cca cca tcc 3206aat agt agc att ctg ttg gat aaa tgt ccg cct cca aga cca cca tcc 3206
Asn Ser Ser Ile Leu Leu Asp Lys Cys Pro Pro Pro Arg Pro Pro SerAsn Ser Ser Ile Leu Leu Asp Lys Cys Pro Pro Pro Arg Pro Pro Ser
910 915 920 925910 915 920 925
tca cca tac cct ccc ttg cca aag gac aag ttg aat cca cct aca cct 3254tca cca tac cct ccc ttg cca aag gac aag ttg aat cca cct aca cct 3254
Ser Pro Tyr Pro Pro Leu Pro Lys Asp Lys Leu Asn Pro Pro Thr ProSer Pro Tyr Pro Pro Leu Pro Lys Asp Lys Leu Asn Pro Pro Thr Pro
930 935 940 930 935 940
agt att tat ttg gaa aat aaa cgt gat gct ttc ttt cct cca tta cat 3302agt att tat ttg gaa aat aaa cgt gat gct ttc ttt cct cca tta cat 3302
Ser Ile Tyr Leu Glu Asn Lys Arg Asp Ala Phe Phe Pro Pro Leu HisSer Ile Tyr Leu Glu Asn Lys Arg Asp Ala Phe Phe Pro Pro Leu His
945 950 955 945 950 955
caa ttt tgt aca aac cca aac aac cct gtt aca gta ata cgt ggc ctt 3350caa ttt tgt aca aac cca aac aac cct gtt aca gta ata cgt ggc ctt 3350
Gln Phe Cys Thr Asn Pro Asn Asn Pro Val Thr Val Ile Arg Gly LeuGln Phe Cys Thr Asn Pro Asn Asn Pro Val Thr Val Ile Arg Gly Leu
960 965 970 960 965 970
gct gga gct ctt aaa tta gac ttg gga ctt ttc tct act aaa act ttg 3398gct gga gct ctt aaa tta gac ttg gga ctt ttc tct act aaa act ttg 3398
Ala Gly Ala Leu Lys Leu Asp Leu Gly Leu Phe Ser Thr Lys Thr LeuAla Gly Ala Leu Lys Leu Asp Leu Gly Leu Phe Ser Thr Lys Thr Leu
975 980 985 975 980 985
gtg gaa gct aac aat gaa cat atg gta gaa gtg agg aca cag ttg tta 3446gtg gaa gct aac aat gaa cat atg gta gaa gtg agg aca cag ttg tta 3446
Val Glu Ala Asn Asn Glu His Met Val Glu Val Arg Thr Gln Leu LeuVal Glu Ala Asn Asn Glu His Met Val Glu Val Arg Thr Gln Leu Leu
990 995 1000 1005990 995 1000 1005
caa cca gca gat gaa aat tgg gac cct act gga acc aag aaa atc 3491caa cca gca gat gaa aat tgg gac cct act gga acc aag aaa atc 3491
Gln Pro Ala Asp Glu Asn Trp Asp Pro Thr Gly Thr Lys Lys IleGln Pro Ala Asp Glu Asn Trp Asp Pro Thr Gly Thr Lys Lys Ile
1010 1015 1020 1010 1015 1020
tgg cac tgt gaa agt aat aga tct cat act aca att gct aaa tat 3536tgg cac tgt gaa agt aat aga tct cat act aca att gct aaa tat 3536
Trp His Cys Glu Ser Asn Arg Ser His Thr Thr Ile Ala Lys TyrTrp His Cys Glu Ser Asn Arg Ser His Thr Thr Ile Ala Lys Tyr
1025 1030 1035 1025 1030 1035
gct cag tac cag gcc tcc tca ttc caa gaa tca ttg aga gaa gaa 3581gct cag tac cag gcc tcc tca ttc caa gaa tca ttg aga gaa gaa 3581
Ala Gln Tyr Gln Ala Ser Ser Phe Gln Glu Ser Leu Arg Glu GluAla Gln Tyr Gln Ala Ser Ser Phe Gln Glu Ser Leu Arg Glu Glu
1040 1045 1050 1040 1045 1050
aat gag aaa aga agt cac cat aaa gac cac tca gac agt gaa tct 3626aat gag aaa aga agt cac cat aaa gac cac tca gac agt gaa tct 3626
Asn Glu Lys Arg Ser His His Lys Asp His Ser Asp Ser Glu SerAsn Glu Lys Arg Ser His His Lys Asp His Ser Asp Ser Glu Ser
1055 1060 1065 1055 1060 1065
aca tca tca gat aat tct ggg aaa aga aga aaa gga ccc ttt aaa 3671aca tca tca gat aat tct ggg aaa aga aga aaa gga ccc ttt aaa 3671
Thr Ser Ser Asp Asn Ser Gly Lys Arg Arg Lys Gly Pro Phe LysThr Ser Ser Asp Asn Ser Gly Lys Arg Arg Lys Gly Pro Phe Lys
1070 1075 1080 1070 1075 1080
acc att aag ttt ggg acc aac att gac ctg tct gat gac aaa aag 3716acc att aag ttt ggg acc aac att gac ctg tct gat gac aaa aag 3716
Thr Ile Lys Phe Gly Thr Asn Ile Asp Leu Ser Asp Asp Lys LysThr Ile Lys Phe Gly Thr Asn Ile Asp Leu Ser Asp Asp Lys Lys
1085 1090 1095 1085 1090 1095
tgg aag tta cag cta cat gag ctg act aaa ctt cct gcc ttc gtg 3761tgg aag tta cag cta cat gag ctg act aaa ctt cct gcc ttc gtg 3761
Trp Lys Leu Gln Leu His Glu Leu Thr Lys Leu Pro Ala Phe ValTrp Lys Leu Gln Leu His Glu Leu Thr Lys Leu Pro Ala Phe Val
1100 1105 1110 1100 1105 1110
aga gtt gta tct gca gga aat ctt tta agc cac gtt ggt cat act 3806aga gtt gta tct gca gga aat ctt tta agc cac gtt ggt cat act 3806
Arg Val Val Ser Ala Gly Asn Leu Leu Ser His Val Gly His ThrArg Val Val Ser Ala Gly Asn Leu Leu Ser His Val Gly His Thr
1115 1120 1125 1115 1120 1125
ata ctg ggc atg aac aca gtt caa cta tac atg aaa gtt cca gga 3851ata ctg ggc atg aac aca gtt caa cta tac atg aaa gtt cca gga 3851
Ile Leu Gly Met Asn Thr Val Gln Leu Tyr Met Lys Val Pro GlyIle Leu Gly Met Asn Thr Val Gln Leu Tyr Met Lys Val Pro Gly
1130 1135 1140 1130 1135 1140
agc aga aca cca ggt cat caa gaa aat aac aac ttc tgt tca gtt 3896agc aga aca cca ggt cat caa gaa aat aac aac ttc tgt tca gtt 3896
Ser Arg Thr Pro Gly His Gln Glu Asn Asn Asn Phe Cys Ser ValSer Arg Thr Pro Gly His Gln Glu Asn Asn Asn Phe Cys Ser Val
1145 1150 1155 1145 1150 1155
aat ata aat att ggc cca ggt gac tgt gaa tgg ttt gtt gtt cct 3941aat ata aat att ggc cca ggt gac tgt gaa tgg ttt gtt gtt cct 3941
Asn Ile Asn Ile Gly Pro Gly Asp Cys Glu Trp Phe Val Val ProAsn Ile Asn Ile Gly Pro Gly Asp Cys Glu Trp Phe Val Val Pro
1160 1165 1170 1160 1165 1170
gaa ggc tac tgg ggt gtt ttg aat gac ttc tgt gaa aaa aat aat 3986gaa ggc tac tgg ggt gtt ttg aat gac ttc tgt gaa aaa aat aat 3986
Glu Gly Tyr Trp Gly Val Leu Asn Asp Phe Cys Glu Lys Asn AsnGlu Gly Tyr Trp Gly Val Leu Asn Asp Phe Cys Glu Lys Asn Asn
1175 1180 1185 1175 1180 1185
ttg aat ttc tta atg ggt tct tgg tgg ccc aac ctt gaa gat cta 4031ttg aat ttc tta atg ggt tct tgg tgg ccc aac ctt gaa gat cta 4031
Leu Asn Phe Leu Met Gly Ser Trp Trp Pro Asn Leu Glu Asp LeuLeu Asn Phe Leu Met Gly Ser Trp Trp Pro Asn Leu Glu Asp Leu
1190 1195 1200 1190 1195 1200
tat gaa gca aat gtt cca gtg tat agg ttt att cag cga cct gga 4076tat gaa gca aat gtt cca gtg tat agg ttt att cag cga cct gga 4076
Tyr Glu Ala Asn Val Pro Val Tyr Arg Phe Ile Gln Arg Pro GlyTyr Glu Ala Asn Val Pro Val Tyr Arg Phe Ile Gln Arg Pro Gly
1205 1210 1215 1205 1210 1215
gat ctg gtc tgg ata aat gct ggc act gtt cat tgg gtt caa gct 4121gat ctg gtc tgg ata aat gct ggc act gtt cat tgg gtt caa gct 4121
Asp Leu Val Trp Ile Asn Ala Gly Thr Val His Trp Val Gln AlaAsp Leu Val Trp Ile Asn Ala Gly Thr Val His Trp Val Gln Ala
1220 1225 1230 1220 1225 1230
att ggc tgg tgc aac aat att gct tgg aat gtt ggt cca ctt aca 4166att ggc tgg tgc aac aat att gct tgg aat gtt ggt cca ctt aca 4166
Ile Gly Trp Cys Asn Asn Ile Ala Trp Asn Val Gly Pro Leu ThrIle Gly Trp Cys Asn Asn Ile Ala Trp Asn Val Gly Pro Leu Thr
1235 1240 1245 1235 1240 1245
gcc tgt cag tat aag tta gca gtg gaa cgt tat gaa tgg aac aag 4211gcc tgt cag tat aag tta gca gtg gaa cgt tat gaa tgg aac aag 4211
Ala Cys Gln Tyr Lys Leu Ala Val Glu Arg Tyr Glu Trp Asn LysAla Cys Gln Tyr Lys Leu Ala Val Glu Arg Tyr Glu Trp Asn Lys
1250 1255 1260 1250 1255 1260
ttg caa aat gta aag tca ata gta ccc atg gtt cat ctt tcc tgg 4256ttg caa aat gta aag tca ata gta ccc atg gtt cat ctt tcc tgg 4256
Leu Gln Asn Val Lys Ser Ile Val Pro Met Val His Leu Ser TrpLeu Gln Asn Val Lys Ser Ile Val Pro Met Val His Leu Ser Trp
1265 1270 1275 1265 1270 1275
aat atg gca cga aat atc aag gtt tca gat cca aag ctt ttt gaa 4301aat atg gca cga aat atc aag gtt tca gat cca aag ctt ttt gaa 4301
Asn Met Ala Arg Asn Ile Lys Val Ser Asp Pro Lys Leu Phe GluAsn Met Ala Arg Asn Ile Lys Val Ser Asp Pro Lys Leu Phe Glu
1280 1285 1290 1280 1285 1290
atg att aag tat tgt ctt ctg aga acg ctg aag caa tgt cag aca 4346atg att aag tat tgt ctt ctg aga acg ctg aag caa tgt cag aca 4346
Met Ile Lys Tyr Cys Leu Leu Arg Thr Leu Lys Gln Cys Gln ThrMet Ile Lys Tyr Cys Leu Leu Arg Thr Leu Lys Gln Cys Gln Thr
1295 1300 1305 1295 1300 1305
ttg agg gaa gct cta att gct gca gga aaa gag atc ata tgg cac 4391ttg agg gaa gct cta att gct gca gga aaa gag atc ata tgg cac 4391
Leu Arg Glu Ala Leu Ile Ala Ala Gly Lys Glu Ile Ile Trp HisLeu Arg Glu Ala Leu Ile Ala Ala Gly Lys Glu Ile Ile Trp His
1310 1315 1320 1310 1315 1320
ggg cgg aca aaa gaa gaa cca gct cat tat tgt agt att tgt gag 4436ggg cgg aca aaa gaa gaa cca gct cat tat tgt agt att tgt gag 4436
Gly Arg Thr Lys Glu Glu Pro Ala His Tyr Cys Ser Ile Cys GluGly Arg Thr Lys Glu Glu Pro Ala His Tyr Cys Ser Ile Cys Glu
1325 1330 1335 1325 1330 1335
gtg gag gtt ttt gat ctg ctt ttt gtc act aat gag agt aat tct 4481gtg gag gtt ttt gat ctg ctt ttt gtc act aat gag agt aat tct 4481
Val Glu Val Phe Asp Leu Leu Phe Val Thr Asn Glu Ser Asn SerVal Glu Val Phe Asp Leu Leu Phe Val Thr Asn Glu Ser Asn Ser
1340 1345 1350 1340 1345 1350
cga aaa acc tac ata gta cat tgc caa gat tgt gca cga aaa aca 4526cga aaa acc tac ata gta cat tgc caa gat tgt gca cga aaa aca 4526
Arg Lys Thr Tyr Ile Val His Cys Gln Asp Cys Ala Arg Lys ThrArg Lys Thr Tyr Ile Val His Cys Gln Asp Cys Ala Arg Lys Thr
1355 1360 1365 1355 1360 1365
agt ggg aat ctg gaa aat ttt gtg gtg cta gaa cag tac aaa atg 4571agt ggg aat ctg gaa aat ttt gtg gtg cta gaa cag tac aaa atg 4571
Ser Gly Asn Leu Glu Asn Phe Val Val Leu Glu Gln Tyr Lys MetSer Gly Asn Leu Glu Asn Phe Val Val Leu Glu Gln Tyr Lys Met
1370 1375 1380 1370 1375 1380
gag gat ctg atg caa gtc tat gac caa ttt aca tta gct cct cca 4616gag gat ctg atg caa gtc tat gac caa ttt aca tta gct cct cca 4616
Glu Asp Leu Met Gln Val Tyr Asp Gln Phe Thr Leu Ala Pro ProGlu Asp Leu Met Gln Val Tyr Asp Gln Phe Thr Leu Ala Pro Pro
1385 1390 1395 1385 1390 1395
tta cca tcc gcc tca tct tga tattgttcca tggacattaa acatgagacc 4667tta cca tcc gcc tca tct tga tattgttcca tggacattaa acatgagacc 4667
Leu Pro Ser Ala Ser SerLeu Pro Ser Ala Ser Ser
1400 1400
ttttctgcta ttcagaaagt aacccagttc tgcaccactg ctatttgtag ctatctcgta 4727ttttctgcta ttcagaaagt aacccagttc tgcaccactg ctatttgtag ctatctcgta 4727
aggctgctgg ctgaaaactg tgtctatgca accttccaag tgtggagtgt caaccaactg 4787aggctgctgg ctgaaaactg tgtctatgca accttccaag tgtggagtgt caaccaactg 4787
gacgggagag tactgctcct atgccaggac tctcgtaaag ctaattagct gaacttcaga 4847gacgggagag tactgctcct atgccaggac tctcgtaaag ctaattagct gaacttcaga 4847
gaaaaaaaaa agaataataa ttaccacgtt ttgtatatat ctgacaaaac tggcaacatc 4907gaaaaaaaaa agaataataa ttaccacgtt ttgtatatat ctgacaaaac tggcaacatc 4907
ataaagacta ctgacttgaa gacaacctct tttatatttc tcgatttctg ggctaatgaa 4967ataaagacta ctgacttgaa gacaacctct tttatatttc tcgatttctg ggctaatgaa 4967
tttgttttca cctatctttt ccctttcaga attttattcg ggggggaaaa taactagcct 5027tttgttttca cctatctttt ccctttcaga attttattcg ggggggaaaa taactagcct 5027
agctggtcat ttctttgtaa ggtagttagc aatatttagt ctttctttgg tcaccttttt 5087agctggtcat ttctttgtaa ggtagttagc aatatttagt ctttctttgg tcaccttttt 5087
ttttttaatg tgaaaagtta agtaagaaac tttttttact gcttttatga atttctgtct 5147ttttttaatg tgaaaagtta agtaagaaac tttttttact gcttttatga atttctgtct 5147
tgttttgaaa ctatgatcgt tataattttg gttcccaaat aaaacattta aaaaaaataa 5207tgttttgaaa ctatgatcgt tataattttg gttcccaaat aaaacattta aaaaaaataa 5207
ttaacagcaa cgtcacaaag ataatggatt gcacatagac taagaaataa acttcagaat 5267ttaacagcaa cgtcacaaag ataatggatt gcacatagac taagaaataa acttcagaat 5267
tgtgattttt tttgtttcta atcttgatgt aaatttacac tatttataaa tacatattta 5327tgtgattttt tttgtttcta atcttgatgt aaatttacac tattattaaa tacatattta 5327
ttgcctgaaa atatttgtga atggaatgct gttatttttt ccagatttac ctgccattga 5387ttgcctgaaa atatttgtga atggaatgct gttatttttt ccagatttac ctgccattga 5387
aattttaagg agttctgtaa tttcaaacac tactcctatt acattttcta tgtgtaaata 5447aattttaagg agttctgtaa tttcaaacac tactcctatt aattttcta tgtgtaaata 5447
aaactgctta gccttgtaca gaaactttta ttaaaattgt ttaatgttta aagagttttc 5507aaactgctta gccttgtaca gaaactttta ttaaaattgt ttaatgttta aagagttttc 5507
tattgtttga gttttaaaaa gaatttatgt acagtgccca atttttgttc acttttcaaa 5567tattgtttga gttttaaaaa gaatttatgt acagtgccca atttttgttc acttttcaaa 5567
tctgattata tatattttat atatacttat gtctgtatat ataatatata tagaaatcta 5627tctgattata tatattttat atatacttat gtctgtatat ataatatata tagaaatcta 5627
gatatatgta taaagattta gaatttgaat ttttcttgtt ttaagtttca catctatggt 5687gatatatgta taaagatta gaatttgaat ttttcttgtt ttaagtttca catctatggt 5687
agatttttga ggtgtctact gtaaagtatt gctcacaaaa agtatgatta tttttaaaga 5747agatttttga ggtgtctact gtaaagtatt gctcacaaaa agtatgatta tttttaaaga 5747
aatatatatg gtatgtatct tcaagaccta aaatgtcaga ctggtttatt gttaaattgc 5807aatatatatg gtatgtatct tcaagaccta aaatgtcaga ctggtttatt gttaaattgc 5807
aactactgca attacagaca aaaaaaatct gccaaatgta gtataataga aaacatagtg 5867aactactgca attacagaca aaaaaaatct gccaaatgta gtataataga aaacatagtg 5867
attgaactta aaactttaaa gggagtttta atacaggctt ttcaaatcct a 5918attgaactta aaactttaaa gggagtttta atacaggctt ttcaaatcct a 5918
<210> 4<210> 4
<211> 1401<211> 1401
<212> PRT<212> PRT
<213> 小鼠(Mus musculus)<213> Mouse (Mus musculus)
<400> 4<400> 4
Met Lys Ser Cys Gly Val Ser Leu Ala Thr Ala Ala Ala Ala Ala AlaMet Lys Ser Cys Gly Val Ser Leu Ala Thr Ala Ala Ala Ala Ala Ala
1 5 10 151 5 10 15
Ala Ala Ala Phe Gly Asp Glu Glu Lys Lys Met Ala Ala Gly Lys AlaAla Ala Ala Phe Gly Asp Glu Glu Lys Lys Met Ala Ala Gly Lys Ala
20 25 30 20 25 30
Ser Gly Glu Ser Glu Glu Ala Ser Pro Ser Leu Thr Ala Glu Glu ArgSer Gly Glu Ser Glu Glu Ala Ser Pro Ser Leu Thr Ala Glu Glu Arg
35 40 45 35 40 45
Glu Ala Leu Gly Gly Leu Asp Ser Arg Leu Phe Gly Phe Val Arg PheGlu Ala Leu Gly Gly Leu Asp Ser Arg Leu Phe Gly Phe Val Arg Phe
50 55 60 50 55 60
His Glu Asp Gly Ala Arg Met Lys Ala Leu Leu Gly Lys Ala Val ArgHis Glu Asp Gly Ala Arg Met Lys Ala Leu Leu Gly Lys Ala Val Arg
65 70 75 8065 70 75 80
Cys Tyr Glu Ser Leu Ile Leu Lys Ala Glu Gly Lys Val Glu Ser AspCys Tyr Glu Ser Leu Ile Leu Lys Ala Glu Gly Lys Val Glu Ser Asp
85 90 95 85 90 95
Phe Phe Cys Gln Leu Gly His Phe Asn Leu Leu Leu Glu Asp Tyr ProPhe Phe Cys Gln Leu Gly His Phe Asn Leu Leu Leu Glu Asp Tyr Pro
100 105 110 100 105 110
Lys Ala Leu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu Gln Ser Asp TyrLys Ala Leu Ser Ala Tyr Gln Arg Tyr Tyr Ser Leu Gln Ser Asp Tyr
115 120 125 115 120 125
Trp Lys Asn Ala Ala Phe Leu Tyr Gly Leu Gly Leu Val Tyr Phe HisTrp Lys Asn Ala Ala Phe Leu Tyr Gly Leu Gly Leu Val Tyr Phe His
130 135 140 130 135 140
Tyr Asn Ala Phe Gln Trp Ala Ile Lys Ala Phe Gln Glu Val Leu TyrTyr Asn Ala Phe Gln Trp Ala Ile Lys Ala Phe Gln Glu Val Leu Tyr
145 150 155 160145 150 155 160
Val Asp Pro Ser Phe Cys Arg Ala Lys Glu Ile His Leu Arg Leu GlyVal Asp Pro Ser Phe Cys Arg Ala Lys Glu Ile His Leu Arg Leu Gly
165 170 175 165 170 175
Leu Met Phe Lys Val Asn Thr Asp Tyr Glu Ser Ser Leu Lys His PheLeu Met Phe Lys Val Asn Thr Asp Tyr Glu Ser Ser Leu Lys His Phe
180 185 190 180 185 190
Gln Leu Ala Leu Val Asp Cys Asn Pro Cys Thr Leu Ser Asn Ala GluGln Leu Ala Leu Val Asp Cys Asn Pro Cys Thr Leu Ser Asn Ala Glu
195 200 205 195 200 205
Ile Gln Phe His Ile Ala His Leu Tyr Glu Thr Gln Arg Lys Tyr HisIle Gln Phe His Ile Ala His Leu Tyr Glu Thr Gln Arg Lys Tyr His
210 215 220 210 215 220
Ser Ala Lys Glu Ala Tyr Glu Gln Leu Leu Gln Thr Glu Asn Leu SerSer Ala Lys Glu Ala Tyr Glu Gln Leu Leu Gln Thr Glu Asn Leu Ser
225 230 235 240225 230 235 240
Ala Gln Val Lys Ala Thr Ile Leu Gln Gln Leu Gly Trp Met His HisAla Gln Val Lys Ala Thr Ile Leu Gln Gln Leu Gly Trp Met His His
245 250 255 245 250 255
Thr Val Asp Leu Leu Gly Asp Lys Ala Thr Lys Glu Ser Tyr Ala IleThr Val Asp Leu Leu Gly Asp Lys Ala Thr Lys Glu Ser Tyr Ala Ile
260 265 270 260 265 270
Gln Tyr Leu Gln Lys Ser Leu Glu Ala Asp Pro Asn Ser Gly Gln SerGln Tyr Leu Gln Lys Ser Leu Glu Ala Asp Pro Asn Ser Gly Gln Ser
275 280 285 275 280 285
Trp Tyr Phe Leu Gly Arg Cys Tyr Ser Ser Ile Gly Lys Val Gln AspTrp Tyr Phe Leu Gly Arg Cys Tyr Ser Ser Ile Gly Lys Val Gln Asp
290 295 300 290 295 300
Ala Phe Ile Ser Tyr Arg Gln Ser Ile Asp Lys Ser Glu Ala Ser AlaAla Phe Ile Ser Tyr Arg Gln Ser Ile Asp Lys Ser Glu Ala Ser Ala
305 310 315 320305 310 315 320
Asp Thr Trp Cys Ser Ile Gly Val Leu Tyr Gln Gln Gln Asn Gln ProAsp Thr Trp Cys Ser Ile Gly Val Leu Tyr Gln Gln Gln Asn Gln Pro
325 330 335 325 330 335
Met Asp Ala Leu Gln Ala Tyr Ile Cys Ala Val Gln Leu Asp His GlyMet Asp Ala Leu Gln Ala Tyr Ile Cys Ala Val Gln Leu Asp His Gly
340 345 350 340 345 350
His Ala Ala Ala Trp Met Asp Leu Gly Thr Leu Tyr Glu Ser Cys AsnHis Ala Ala Ala Trp Met Asp Leu Gly Thr Leu Tyr Glu Ser Cys Asn
355 360 365 355 360 365
Gln Pro Gln Asp Ala Ile Lys Cys Tyr Leu Asn Ala Thr Arg Ser LysGln Pro Gln Asp Ala Ile Lys Cys Tyr Leu Asn Ala Thr Arg Ser Lys
370 375 380 370 375 380
Asn Cys Ser Asn Thr Ser Gly Leu Ala Ala Arg Ile Lys Tyr Leu GlnAsn Cys Ser Asn Thr Ser Gly Leu Ala Ala Arg Ile Lys Tyr Leu Gln
385 390 395 400385 390 395 400
Ala Gln Leu Cys Asn Leu Pro Gln Gly Ser Leu Gln Asn Lys Thr LysAla Gln Leu Cys Asn Leu Pro Gln Gly Ser Leu Gln Asn Lys Thr Lys
405 410 415 405 410 415
Leu Leu Pro Ser Ile Glu Glu Ala Trp Ser Leu Pro Ile Pro Ala GluLeu Leu Pro Ser Ile Glu Glu Ala Trp Ser Leu Pro Ile Pro Ala Glu
420 425 430 420 425 430
Leu Thr Ser Arg Gln Gly Ala Met Asn Thr Ala Gln Gln Asn Thr SerLeu Thr Ser Arg Gln Gly Ala Met Asn Thr Ala Gln Gln Asn Thr Ser
435 440 445 435 440 445
Asp Asn Trp Ser Gly Gly Asn Ala Pro Pro Pro Val Glu Gln Gln ThrAsp Asn Trp Ser Gly Gly Asn Ala Pro Pro Pro Val Glu Gln Gln Thr
450 455 460 450 455 460
His Ser Trp Cys Leu Thr Pro Gln Lys Leu Gln His Leu Glu Gln LeuHis Ser Trp Cys Leu Thr Pro Gln Lys Leu Gln His Leu Glu Gln Leu
465 470 475 480465 470 475 480
Arg Ala Asn Arg Asn Asn Leu Asn Pro Ala Gln Lys Leu Met Leu GluArg Ala Asn Arg Asn Asn Leu Asn Pro Ala Gln Lys Leu Met Leu Glu
485 490 495 485 490 495
Gln Leu Glu Ser Gln Phe Val Leu Met Gln Gln His Gln Met Arg GlnGln Leu Glu Ser Gln Phe Val Leu Met Gln Gln His Gln Met Arg Gln
500 505 510 500 505 510
Thr Gly Val Ala Gln Val Arg Pro Thr Gly Ile Leu Asn Gly Pro ThrThr Gly Val Ala Gln Val Arg Pro Thr Gly Ile Leu Asn Gly Pro Thr
515 520 525 515 520 525
Val Asp Ser Ser Leu Pro Thr Asn Ser Val Ser Gly Gln Gln Pro GlnVal Asp Ser Ser Leu Pro Thr Asn Ser Val Ser Gly Gln Gln Pro Gln
530 535 540 530 535 540
Leu Pro Leu Thr Arg Met Pro Ser Val Ser Gln Pro Gly Val His ThrLeu Pro Leu Thr Arg Met Pro Ser Val Ser Gln Pro Gly Val His Thr
545 550 555 560545 550 555 560
Ala Cys Pro Arg Gln Thr Leu Ala Asn Gly Pro Phe Ser Ala Gly HisAla Cys Pro Arg Gln Thr Leu Ala Asn Gly Pro Phe Ser Ala Gly His
565 570 575 565 570 575
Val Pro Cys Ser Thr Ser Arg Thr Leu Gly Ser Thr Asp Thr Val LeuVal Pro Cys Ser Thr Ser Arg Thr Leu Gly Ser Thr Asp Thr Val Leu
580 585 590 580 585 590
Ile Gly Asn Asn His Val Thr Gly Ser Gly Ser Asn Gly Asn Val ProIle Gly Asn Asn His Val Thr Gly Ser Gly Ser Asn Gly Asn Val Pro
595 600 605 595 600 605
Tyr Leu Gln Arg Asn Ala Pro Thr Leu Pro His Asn Arg Thr Asn LeuTyr Leu Gln Arg Asn Ala Pro Thr Leu Pro His Asn Arg Thr Asn Leu
610 615 620 610 615 620
Thr Ser Ser Thr Glu Glu Pro Trp Lys Asn Gln Leu Ser Asn Ser ThrThr Ser Ser Thr Glu Glu Pro Trp Lys Asn Gln Leu Ser Asn Ser Thr
625 630 635 640625 630 635 640
Gln Gly Leu His Lys Gly Pro Ser Ser His Leu Ala Gly Pro Asn GlyGln Gly Leu His Lys Gly Pro Ser Ser His Leu Ala Gly Pro Asn Gly
645 650 655 645 650 655
Glu Arg Pro Leu Ser Ser Thr Gly Pro Ser Gln His Leu Gln Ala AlaGlu Arg Pro Leu Ser Ser Thr Gly Pro Ser Gln His Leu Gln Ala Ala
660 665 670 660 665 670
Gly Ser Gly Ile Gln Asn Gln Asn Gly His Pro Thr Leu Pro Ser AsnGly Ser Gly Ile Gln Asn Gln Asn Gly His Pro Thr Leu Pro Ser Asn
675 680 685 675 680 685
Ser Val Thr Gln Gly Ala Ala Leu Asn His Leu Ser Ser His Thr AlaSer Val Thr Gln Gly Ala Ala Leu Asn His Leu Ser Ser His Thr Ala
690 695 700 690 695 700
Thr Ser Gly Gly Gln Gln Gly Ile Thr Leu Thr Lys Glu Ser Lys ProThr Ser Gly Gly Gln Gln Gly Ile Thr Leu Thr Lys Glu Ser Lys Pro
705 710 715 720705 710 715 720
Ser Gly Asn Thr Leu Thr Val Pro Glu Thr Ser Arg Gln Thr Gly GluSer Gly Asn Thr Leu Thr Val Pro Glu Thr Ser Arg Gln Thr Gly Glu
725 730 735 725 730 735
Thr Pro Asn Ser Thr Ala Ser Val Glu Gly Leu Pro Asn His Val HisThr Pro Asn Ser Thr Ala Ser Val Glu Gly Leu Pro Asn His Val His
740 745 750 740 745 750
Gln Val Met Ala Asp Ala Val Cys Ser Pro Ser His Gly Asp Ser LysGln Val Met Ala Asp Ala Val Cys Ser Pro Ser His Gly Asp Ser Lys
755 760 765 755 760 765
Ser Pro Gly Leu Leu Ser Ser Asp Asn Pro Gln Leu Ser Ala Leu LeuSer Pro Gly Leu Leu Ser Ser Asp Asn Pro Gln Leu Ser Ala Leu Leu
770 775 780 770 775 780
Met Gly Lys Ala Asn Asn Asn Val Gly Pro Gly Thr Cys Asp Lys ValMet Gly Lys Ala Asn Asn Asn Val Gly Pro Gly Thr Cys Asp Lys Val
785 790 795 800785 790 795 800
Asn Asn Ile His Pro Thr Val His Thr Lys Thr Asp Asn Ser Val AlaAsn Asn Ile His Pro Thr Val His Thr Lys Thr Asp Asn Ser Val Ala
805 810 815 805 810 815
Ser Ser Pro Ser Ser Ala Ile Ser Thr Ala Thr Pro Ser Pro Lys SerSer Ser Pro Ser Ser Ala Ile Ser Thr Ala Thr Pro Ser Pro Lys Ser
820 825 830 820 825 830
Thr Glu Gln Thr Thr Thr Asn Ser Val Thr Ser Leu Asn Ser Pro HisThr Glu Gln Thr Thr Thr Asn Ser Val Thr Ser Leu Asn Ser Pro His
835 840 845 835 840 845
Ser Gly Leu His Thr Ile Asn Gly Glu Gly Met Glu Glu Ser Gln SerSer Gly Leu His Thr Ile Asn Gly Glu Gly Met Glu Glu Ser Gln Ser
850 855 860 850 855 860
Pro Ile Lys Thr Asp Leu Leu Leu Val Ser His Arg Pro Ser Pro GlnPro Ile Lys Thr Asp Leu Leu Leu Val Ser His Arg Pro Ser Pro Gln
865 870 875 880865 870 875 880
Ile Ile Pro Ser Met Ser Val Ser Ile Tyr Pro Ser Ser Ala Glu ValIle Ile Pro Ser Met Ser Val Ser Ile Tyr Pro Ser Ser Ala Glu Val
885 890 895 885 890 895
Leu Lys Ala Cys Arg Asn Leu Gly Lys Asn Gly Leu Ser Asn Ser SerLeu Lys Ala Cys Arg Asn Leu Gly Lys Asn Gly Leu Ser Asn Ser Ser
900 905 910 900 905 910
Ile Leu Leu Asp Lys Cys Pro Pro Pro Arg Pro Pro Ser Ser Pro TyrIle Leu Leu Asp Lys Cys Pro Pro Pro Arg Pro Pro Ser Ser Pro Tyr
915 920 925 915 920 925
Pro Pro Leu Pro Lys Asp Lys Leu Asn Pro Pro Thr Pro Ser Ile TyrPro Pro Leu Pro Lys Asp Lys Leu Asn Pro Pro Thr Pro Ser Ile Tyr
930 935 940 930 935 940
Leu Glu Asn Lys Arg Asp Ala Phe Phe Pro Pro Leu His Gln Phe CysLeu Glu Asn Lys Arg Asp Ala Phe Phe Pro Pro Leu His Gln Phe Cys
945 950 955 960945 950 955 960
Thr Asn Pro Asn Asn Pro Val Thr Val Ile Arg Gly Leu Ala Gly AlaThr Asn Pro Asn Asn Pro Val Thr Val Ile Arg Gly Leu Ala Gly Ala
965 970 975 965 970 975
Leu Lys Leu Asp Leu Gly Leu Phe Ser Thr Lys Thr Leu Val Glu AlaLeu Lys Leu Asp Leu Gly Leu Phe Ser Thr Lys Thr Leu Val Glu Ala
980 985 990 980 985 990
Asn Asn Glu His Met Val Glu Val Arg Thr Gln Leu Leu Gln Pro AlaAsn Asn Glu His Met Val Glu Val Arg Thr Gln Leu Leu Gln Pro Ala
995 1000 1005 995 1000 1005
Asp Glu Asn Trp Asp Pro Thr Gly Thr Lys Lys Ile Trp His CysAsp Glu Asn Trp Asp Pro Thr Gly Thr Lys Lys Ile Trp His Cys
1010 1015 1020 1010 1015 1020
Glu Ser Asn Arg Ser His Thr Thr Ile Ala Lys Tyr Ala Gln TyrGlu Ser Asn Arg Ser His Thr Thr Ile Ala Lys Tyr Ala Gln Tyr
1025 1030 1035 1025 1030 1035
Gln Ala Ser Ser Phe Gln Glu Ser Leu Arg Glu Glu Asn Glu LysGln Ala Ser Ser Phe Gln Glu Ser Leu Arg Glu Glu Asn Glu Lys
1040 1045 1050 1040 1045 1050
Arg Ser His His Lys Asp His Ser Asp Ser Glu Ser Thr Ser SerArg Ser His His Lys Asp His Ser Asp Ser Glu Ser Thr Ser Ser
1055 1060 1065 1055 1060 1065
Asp Asn Ser Gly Lys Arg Arg Lys Gly Pro Phe Lys Thr Ile LysAsp Asn Ser Gly Lys Arg Arg Lys Gly Pro Phe Lys Thr Ile Lys
1070 1075 1080 1070 1075 1080
Phe Gly Thr Asn Ile Asp Leu Ser Asp Asp Lys Lys Trp Lys LeuPhe Gly Thr Asn Ile Asp Leu Ser Asp Asp Lys Lys Trp Lys Leu
1085 1090 1095 1085 1090 1095
Gln Leu His Glu Leu Thr Lys Leu Pro Ala Phe Val Arg Val ValGln Leu His Glu Leu Thr Lys Leu Pro Ala Phe Val Arg Val Val
1100 1105 1110 1100 1105 1110
Ser Ala Gly Asn Leu Leu Ser His Val Gly His Thr Ile Leu GlySer Ala Gly Asn Leu Leu Ser His Val Gly His Thr Ile Leu Gly
1115 1120 1125 1115 1120 1125
Met Asn Thr Val Gln Leu Tyr Met Lys Val Pro Gly Ser Arg ThrMet Asn Thr Val Gln Leu Tyr Met Lys Val Pro Gly Ser Arg Thr
1130 1135 1140 1130 1135 1140
Pro Gly His Gln Glu Asn Asn Asn Phe Cys Ser Val Asn Ile AsnPro Gly His Gln Glu Asn Asn Asn Phe Cys Ser Val Asn Ile Asn
1145 1150 1155 1145 1150 1155
Ile Gly Pro Gly Asp Cys Glu Trp Phe Val Val Pro Glu Gly TyrIle Gly Pro Gly Asp Cys Glu Trp Phe Val Val Pro Glu Gly Tyr
1160 1165 1170 1160 1165 1170
Trp Gly Val Leu Asn Asp Phe Cys Glu Lys Asn Asn Leu Asn PheTrp Gly Val Leu Asn Asp Phe Cys Glu Lys Asn Asn Leu Asn Phe
1175 1180 1185 1175 1180 1185
Leu Met Gly Ser Trp Trp Pro Asn Leu Glu Asp Leu Tyr Glu AlaLeu Met Gly Ser Trp Trp Pro Asn Leu Glu Asp Leu Tyr Glu Ala
1190 1195 1200 1190 1195 1200
Asn Val Pro Val Tyr Arg Phe Ile Gln Arg Pro Gly Asp Leu ValAsn Val Pro Val Tyr Arg Phe Ile Gln Arg Pro Gly Asp Leu Val
1205 1210 1215 1205 1210 1215
Trp Ile Asn Ala Gly Thr Val His Trp Val Gln Ala Ile Gly TrpTrp Ile Asn Ala Gly Thr Val His Trp Val Gln Ala Ile Gly Trp
1220 1225 1230 1220 1225 1230
Cys Asn Asn Ile Ala Trp Asn Val Gly Pro Leu Thr Ala Cys GlnCys Asn Asn Ile Ala Trp Asn Val Gly Pro Leu Thr Ala Cys Gln
1235 1240 1245 1235 1240 1245
Tyr Lys Leu Ala Val Glu Arg Tyr Glu Trp Asn Lys Leu Gln AsnTyr Lys Leu Ala Val Glu Arg Tyr Glu Trp Asn Lys Leu Gln Asn
1250 1255 1260 1250 1255 1260
Val Lys Ser Ile Val Pro Met Val His Leu Ser Trp Asn Met AlaVal Lys Ser Ile Val Pro Met Val His Leu Ser Trp Asn Met Ala
1265 1270 1275 1265 1270 1275
Arg Asn Ile Lys Val Ser Asp Pro Lys Leu Phe Glu Met Ile LysArg Asn Ile Lys Val Ser Asp Pro Lys Leu Phe Glu Met Ile Lys
1280 1285 1290 1280 1285 1290
Tyr Cys Leu Leu Arg Thr Leu Lys Gln Cys Gln Thr Leu Arg GluTyr Cys Leu Leu Arg Thr Leu Lys Gln Cys Gln Thr Leu Arg Glu
1295 1300 1305 1295 1300 1305
Ala Leu Ile Ala Ala Gly Lys Glu Ile Ile Trp His Gly Arg ThrAla Leu Ile Ala Ala Gly Lys Glu Ile Ile Trp His Gly Arg Thr
1310 1315 1320 1310 1315 1320
Lys Glu Glu Pro Ala His Tyr Cys Ser Ile Cys Glu Val Glu ValLys Glu Glu Pro Ala His Tyr Cys Ser Ile Cys Glu Val Glu Val
1325 1330 1335 1325 1330 1335
Phe Asp Leu Leu Phe Val Thr Asn Glu Ser Asn Ser Arg Lys ThrPhe Asp Leu Leu Phe Val Thr Asn Glu Ser Asn Ser Arg Lys Thr
1340 1345 1350 1340 1345 1350
Tyr Ile Val His Cys Gln Asp Cys Ala Arg Lys Thr Ser Gly AsnTyr Ile Val His Cys Gln Asp Cys Ala Arg Lys Thr Ser Gly Asn
1355 1360 1365 1355 1360 1365
Leu Glu Asn Phe Val Val Leu Glu Gln Tyr Lys Met Glu Asp LeuLeu Glu Asn Phe Val Val Leu Glu Gln Tyr Lys Met Glu Asp Leu
1370 1375 1380 1370 1375 1380
Met Gln Val Tyr Asp Gln Phe Thr Leu Ala Pro Pro Leu Pro SerMet Gln Val Tyr Asp Gln Phe Thr Leu Ala Pro Pro Leu Pro Ser
1385 1390 1395 1385 1390 1395
Ala Ser SerAla Ser Ser
1400 1400
<210> 5<210> 5
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequence
<400> 5<400> 5
gcugcuacga aucucuaau 19gcugcuacga auccuaau 19
<210> 6<210> 6
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequence
<400> 6<400> 6
auuagagauu cguagcagc 19auuagagauu cguagcagc 19
<210> 7<210> 7
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequence
<400> 7<400> 7
uucuccgaac gugucacgu 19uucuccgaac gugucacgu 19
<210> 8<210> 8
<211> 19<211> 19
<212> RNA<212> RNA
<213> 人工序列<213> Artificial sequence
<400> 8<400> 8
acgugacacg uucggagaa 19acgugacacg uucggagaa 19
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710116926.0ACN107096015A (en) | 2017-03-01 | 2017-03-01 | The antivirus action of novel antiviral molecule Kdm6a a kind of and its application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710116926.0ACN107096015A (en) | 2017-03-01 | 2017-03-01 | The antivirus action of novel antiviral molecule Kdm6a a kind of and its application |
| Publication Number | Publication Date |
|---|---|
| CN107096015Atrue CN107096015A (en) | 2017-08-29 |
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| CN201710116926.0APendingCN107096015A (en) | 2017-03-01 | 2017-03-01 | The antivirus action of novel antiviral molecule Kdm6a a kind of and its application |
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| CN (1) | CN107096015A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109550051A (en)* | 2019-01-31 | 2019-04-02 | 上海交通大学医学院附属第九人民医院 | Histone demethylase KDM6A inhibitor is in the purposes for preparing obesity treatment drugs |
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