CN111424017B - Exosome loading shRNA (short hairpin ribonucleic acid) and construction method and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种装载shRNA的外泌体及其构建方法与应用。本发明通过将可以有效沉默靶基因的shRNA序列克隆入表达载体，得到重组表达载体1；shRNA序列包括依次连接的靶序列、茎环结构序列和靶序列的互补序列；然后将能与所述的茎环结构特异性结合的蛋白A的基因与外泌体膜蛋白的基因连接，得到融合基因序列，再将所得的融合基因序列克隆入表达载体，得到重组表达载体2；最后将重组表达载体1、2同时转染细胞，培养转染后细胞，收集外泌体，即获得所述的装载shRNA的外泌体。该方法操作简单易行，工艺稳定，适用性好。所得外泌体中包载有大量shRNA，可以作为一种高效的药物传递系统。

The invention discloses an exosome loaded with shRNA and a construction method and application thereof. In the present invention, the recombinant expression vector 1 is obtained by cloning the shRNA sequence that can effectively silence the target gene into the expression vector; the shRNA sequence includes the target sequence, the stem-loop structure sequence and the complementary sequence of the target sequence connected in sequence; The gene of the protein A specifically bound by the stem-loop structure is connected with the gene of the exosome membrane protein to obtain a fusion gene sequence, and then the obtained fusion gene sequence is cloned into an expression vector to obtain a recombinant expression vector 2; finally, the recombinant expression vector 1 , 2 Simultaneously transfect cells, culture the transfected cells, and collect exosomes, that is, to obtain the shRNA-loaded exosomes. The method is simple and easy to operate, has stable process and good applicability. The obtained exosomes contain a large amount of shRNA, which can be used as an efficient drug delivery system.

Description

Translated fromChinese

一种装载shRNA的外泌体及其构建方法与应用A kind of exosome loaded with shRNA and its construction method and application

技术领域technical field

本发明属于生物技术领域，特别涉及一种装载shRNA的外泌体及其构建方法与应用。The invention belongs to the field of biotechnology, and particularly relates to an exosome loaded with shRNA and a construction method and application thereof.

背景技术Background technique

RNA干扰(RNAi)是有效沉默或抑制目标基因表达的过程，该过程通过双链RNA(dsRNA)使得目标基因相应的mRNA选择性失活来实现的。沉默机制可导致由小干扰RNA(siRNA)或短发夹RNA(shRNA)诱导实现靶mRNA的降解，或者通过小RNA(miRNA)诱导特定mRNA翻译的抑制。siRNA和shRNA都已用来基因治疗。其中短发夹RNA(shRNA)，包含一个环结构，可加工成siRNA发挥作用，也可通过与目标mRNA互补结合序列特异性地实现靶mRNA降解。shRNA比siRNA的优势包括能够使用病毒载体进行转染，克服了某些类型的细胞不能转染的难题，能选择使用诱导型启动子控制shRNA表达，能够与报告基因共表达。此外，它们能减少脱靶效应。RNA interference (RNAi) is the process of effectively silencing or inhibiting the expression of target genes by selectively inactivating the corresponding mRNAs of target genes by double-stranded RNA (dsRNA). Silencing mechanisms can lead to degradation of target mRNAs induced by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), or inhibition of translation of specific mRNAs induced by small RNAs (miRNAs). Both siRNA and shRNA have been used in gene therapy. Among them, short hairpin RNA (shRNA), which contains a loop structure, can be processed into siRNA to function, and can also specifically degrade target mRNA through a complementary binding sequence to the target mRNA. The advantages of shRNA over siRNA include the ability to use viral vectors for transfection, overcoming the inability to transfect certain types of cells, the option to use inducible promoters to control shRNA expression, and the ability to co-express with reporter genes. Furthermore, they reduce off-target effects.

外泌体(exosomes)是由细胞分泌的直径为30～150nm的囊泡，细胞质膜凹陷形成的细胞内小泡，具有与细胞膜相同的膜结构，可以携带诸如RNA、蛋白质等大量成分，与细胞的生物学功能及细胞间的信号传递有着密切的关系。因其特殊的结构，相较人工合成的药物载体，外泌体作为药物载体进行药物运输有独特的优势，主要体现在外泌体的膜结构与细胞膜相同，可以提高药物进入细胞的效率，甚至可以透过血脑屏障，同时外泌体引起的有害免疫反应极低，有很好的渗透滞留(EPR)效应从而具有缓释效果等。目前已经尝试用外泌体携带siRNA、化学小分子药物等进行基因治疗和肿瘤治疗等研究。外泌体包载siRNA等小分子多采用将外泌体与小分子孵育的方式，包载率一般较低。Exosomes are vesicles with a diameter of 30 to 150 nm secreted by cells, intracellular vesicles formed by the depression of the cytoplasmic membrane, with the same membrane structure as the cell membrane, and can carry a large number of components such as RNA and protein. There is a close relationship between the biological function and the signal transmission between cells. Due to its special structure, compared with synthetic drug carriers, exosomes have unique advantages as drug carriers for drug transport, mainly in that the membrane structure of exosomes is the same as that of cell membranes, which can improve the efficiency of drug entry into cells, and even Through the blood-brain barrier, the harmful immune response caused by exosomes is extremely low, and it has a good osmotic retention (EPR) effect and has a sustained release effect. At present, attempts have been made to use exosomes to carry siRNA, chemical small molecule drugs, etc. for gene therapy and tumor therapy. Small molecules such as siRNA encapsulated in exosomes are mostly incubated with exosomes and small molecules, and the encapsulation rate is generally low.

因此，如何实现外泌体对shRNA更高效的包载，是当前亟待解决研究的关键问题。Therefore, how to achieve more efficient encapsulation of shRNA by exosomes is a key problem that needs to be solved urgently.

发明内容SUMMARY OF THE INVENTION

本发明的首要目的在于克服现有技术的缺点与不足，提供一种装载shRNA的外泌体的构建方法。The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a method for constructing shRNA-loaded exosomes.

本发明的另一目的在于提供一种装载shRNA的外泌体。Another object of the present invention is to provide an exosome loaded with shRNA.

本发明的另一目的在于提供上述装载shRNA的外泌体的应用。Another object of the present invention is to provide the application of the above-mentioned shRNA-loaded exosomes.

本发明的目的通过下述技术方案实现：The object of the present invention is achieved through the following technical solutions:

一种装载shRNA的外泌体的构建方法，包括如下步骤：A method for constructing exosomes loaded with shRNA, comprising the following steps:

(1)设计、合成可以有效沉默靶基因的shRNA序列；将所述的shRNA序列克隆入表达载体，得到重组表达载体1；所述的shRNA序列包括依次连接的靶序列、茎环结构(Kt-loop)序列和靶序列的互补序列；(1) Design and synthesize an shRNA sequence that can effectively silence the target gene; clone the shRNA sequence into an expression vector to obtain arecombinant expression vector 1; the shRNA sequence includes the target sequence, stem-loop structure (Kt- loop) sequence and the complementary sequence of the target sequence;

(2)将能与所述的茎环结构特异性结合的蛋白的基因与外泌体膜蛋白的基因连接，得到融合基因序列，再将所得的融合基因序列克隆入表达载体，得到重组表达载体2；(2) connecting the gene of the protein that can specifically bind to the stem-loop structure with the gene of the exosome membrane protein to obtain a fusion gene sequence, and then clone the obtained fusion gene sequence into an expression vector to obtain arecombinant expression vector 2;

(3)将步骤(1)得到的重组表达载体1和步骤(2)得到的重组表达载体2同时转染细胞，培养转染后细胞，收集外泌体，即获得所述的装载shRNA的外泌体。(3) Simultaneously transfect the cells with therecombinant expression vector 1 obtained in step (1) and therecombinant expression vector 2 obtained in step (2), culture the transfected cells, and collect exosomes to obtain the exosomes loaded with shRNA. exosomes.

设计步骤(1)中所述的shRNA序列时，需通过构建包含所述的shRNA序列的重组表达载体，转染细胞，验证其具有沉默靶基因的功能。When designing the shRNA sequence described in step (1), it is necessary to construct a recombinant expression vector containing the shRNA sequence, transfect cells, and verify that it has the function of silencing the target gene.

步骤(1)中所述的茎环结构序列为GCTGACCCGAAAGGGCGTGATGC。The stem-loop structure sequence described in step (1) is GCTGACCCGAAAGGGCGTGATGC.

步骤(1)中所述的表达载体优选为pRNAT-U6.1/Neo。The expression vector described in step (1) is preferably pRNAT-U6.1/Neo.

步骤(2)中所述的能与茎环结构特异性结合的蛋白可选自RNA结合蛋白L7Ae、NHP2核糖核蛋白样蛋白1(NH2L1_HUMAN NHP2-like protein 1)、人核蛋白P56(HumanNOP56_HUMAN Nucleolar protein 56)和人核蛋白P58(HumanNOP58_HUMAN Nucleolar protein58)中的至少一种；优选为RNA结合蛋白L7Ae。The protein that can specifically bind to the stem-loop structure described in step (2) can be selected from RNA-binding protein L7Ae, NHP2 ribonucleoprotein-like protein 1 (NH2L1_HUMAN NHP2-like protein 1), human nucleoprotein P56 (HumanNOP56_HUMAN Nucleolar protein 56) and at least one of human nucleoprotein P58 (HumanNOP58_HUMAN Nucleolar protein58); preferably RNA binding protein L7Ae.

所述的RNA结合蛋白L7Ae的氨基酸序列如SEQ ID NO.3所示。The amino acid sequence of the RNA binding protein L7Ae is shown in SEQ ID NO.3.

所述的RNA结合蛋白L7Ae的核苷酸序列如SEQ ID NO.4所示。The nucleotide sequence of the RNA binding protein L7Ae is shown in SEQ ID NO.4.

所述的NHP2核糖核蛋白样蛋白1的氨基酸序列如SEQ ID NO.5所示。The amino acid sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO.5.

所述的NHP2核糖核蛋白样蛋白1的核苷酸序列如SEQ ID NO.6所示。The nucleotide sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO.6.

所述的人核蛋白P56的氨基酸序列如SEQ ID NO.7所示。The amino acid sequence of the human nucleoprotein P56 is shown in SEQ ID NO.7.

所述的人核蛋白P56的核苷酸序列如SEQ ID NO.8所示。The nucleotide sequence of the human nucleoprotein P56 is shown in SEQ ID NO.8.

所述的人核蛋白P58的氨基酸序列如SEQ ID NO.9所示。The amino acid sequence of the human nucleoprotein P58 is shown in SEQ ID NO.9.

所述的人核蛋白P58的核苷酸序列如SEQ ID NO.10所示。The nucleotide sequence of the human nucleoprotein P58 is shown in SEQ ID NO.10.

步骤(2)中所述的外泌体膜蛋白优选为CD63、CD81、CD9和TP01中的至少一种；更优选为CD63。The exosomal membrane protein described in step (2) is preferably at least one of CD63, CD81, CD9 and TP01; more preferably CD63.

所述的CD63的氨基酸序列如SEQ ID NO.11所示。The amino acid sequence of CD63 is shown in SEQ ID NO.11.

所述的CD63的核苷酸序列如SEQ ID NO.12所示。The nucleotide sequence of CD63 is shown in SEQ ID NO.12.

所述的CD81的氨基酸序列如SEQ ID NO.13所示。The amino acid sequence of CD81 is shown in SEQ ID NO.13.

所述的CD81的核苷酸序列如SEQ ID NO.14所示。The nucleotide sequence of CD81 is shown in SEQ ID NO.14.

所述的CD9的氨基酸序列如SEQ ID NO.15所示。The amino acid sequence of CD9 is shown in SEQ ID NO.15.

所述的CD9的核苷酸序列如SEQ ID NO.16所示。The nucleotide sequence of CD9 is shown in SEQ ID NO.16.

所述的TP01的氨基酸序列如SEQ ID NO.17所示。The amino acid sequence of TP01 is shown in SEQ ID NO.17.

所述的TP01的核苷酸序列如SEQ ID NO.18所示。The nucleotide sequence of TP01 is shown in SEQ ID NO.18.

步骤(2)中所述的连接优选为通过接头连接，连接的顺序为蛋白A的基因片段-接头-外泌体膜蛋白基因片段。The connection described in step (2) is preferably connected by a linker, and the sequence of linking is protein A gene fragment-linker-exosome membrane protein gene fragment.

所述的接头的核苷酸序列为：The nucleotide sequence of the linker is:

GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC。GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC.

步骤(2)中所述的表达载体优选为pCDNA3.4。The expression vector described in step (2) is preferably pCDNA3.4.

步骤(3)中，在转染细胞时，重组表达载体1和重组表达载体2的数量比优选为1～2:1～2；更优选为1:1。In step (3), when transfecting cells, the quantity ratio ofrecombinant expression vector 1 andrecombinant expression vector 2 is preferably 1-2:1-2; more preferably 1:1.

步骤(3)中，收集外泌体的方法参照中国专利申请CN201811023079.4实施例2步骤3中“外泌体的提取”的方法、步骤进行。In step (3), the method for collecting exosomes is carried out with reference to the method and steps of "extraction of exosomes" instep 3 of Example 2 of Chinese Patent Application CN201811023079.4.

所述的装载shRNA的外泌体的构建方法可适用于多种靶基因。所述的靶基因可以根据需要进行选择，例如EGFP、bFGF，EGFR等。The described method for constructing shRNA-loaded exosomes can be applied to a variety of target genes. The target gene can be selected according to needs, such as EGFP, bFGF, EGFR and so on.

当所述的靶基因为EGFP时，所述的靶序列为GGCATCAAGGTGAACTTCA。When the target gene is EGFP, the target sequence is GGCATCAAGGTGAACTTCA.

一种装载shRNA的外泌体，通过上述构建方法得到。An exosome loaded with shRNA is obtained by the above construction method.

上述外泌体在作为基因治疗的靶向递送系统中的应用。Use of the above exosomes as a targeted delivery system for gene therapy.

本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:

1.本发明提供了一种装载shRNA的外泌体的构建方法。本发明利用shRNA特殊的发夹结构(Kt-环)，以及可以与Kt-环特异性结合的蛋白，通过质粒转染的方法有目的地将shRNA拉进外泌体中，从而建立一种新的shRNA载入外泌体的方法。该方法可显著提高shRNA的包载效率，提高外泌体的载药量，操作简单易行，工艺稳定，适用性好。1. The present invention provides a method for constructing shRNA-loaded exosomes. The present invention utilizes the special hairpin structure (Kt-loop) of shRNA and a protein that can specifically bind to Kt-loop, and purposefully pulls shRNA into exosomes through plasmid transfection, thereby establishing a new A method for loading shRNA into exosomes. The method can significantly improve the encapsulation efficiency of shRNA, increase the drug-loading capacity of exosomes, and is easy to operate, stable in process, and good in applicability.

2.本发明还提供了一种装载shRNA的外泌体，该外泌体中包载有大量shRNA，外泌体能通过膜融合的方式将shRNA释放到受体细胞中，可以作为一种高效的药物传递系统，应用于基因治疗和肿瘤治疗等研究中。2. The present invention also provides an exosome loaded with shRNA, the exosome contains a large amount of shRNA, and the exosome can release the shRNA into the recipient cells by means of membrane fusion, which can be used as an efficient Drug delivery system, used in gene therapy and tumor therapy research.

附图说明Description of drawings

图1为实施例1中shRNA沉默EGFP的细胞荧光结果照片图，其中，A为未转染质粒的细胞，B为同时转染pcDNA3.4-EGFP质粒和pRNAT-U6.1-sh-N质粒的细胞，C为同时转染pcDNA3.4-EGFP质粒和pRNAT-U6.1-Kt-shEGFP质粒的细胞。Figure 1 is a photo of the cytofluorescence results of shRNA silencing EGFP in Example 1, wherein A is a cell without plasmid transfection, and B is a plasmid transfected with pcDNA3.4-EGFP and pRNAT-U6.1-sh-N at the same time The cells in C are cells transfected with pcDNA3.4-EGFP plasmid and pRNAT-U6.1-Kt-shEGFP plasmid at the same time.

图2为外泌体的NTA检测结果分析图。Figure 2 is an analysis diagram of NTA detection results of exosomes.

图3为外泌体中标志蛋白的Western blot鉴定结果图，其中，泳道1为Kt-shEGFP+pCDNA3.4组，泳道2为Kt-shEGFP+CD63-L7Ae组。Figure 3 shows the results of Western blot identification of marker proteins in exosomes.Lane 1 is the Kt-shEGFP+pCDNA3.4 group, andlane 2 is the Kt-shEGFP+CD63-L7Ae group.

图4为实施例6中外泌体中shRNA的检测结果分析图。4 is an analysis diagram of the detection results of shRNA in exosomes in Example 6.

图5为实施例7中不同处理组对EGFP的表达的Western blot检测结果图，其中，泳道1-3为对照组，泳道4-6为Kt-shEGFP+CD63-L7Ae组，泳道7-9为Kt-shEGFP+pCDNA3.4组。Figure 5 is the Western blot detection result of EGFP expression in different treatment groups in Example 7, wherein, lanes 1-3 are the control group, lanes 4-6 are the Kt-shEGFP+CD63-L7Ae group, and lanes 7-9 are the Kt-shEGFP+pCDNA3.4 group.

图6为实施例7中不同处理组对EGFP的表达的Western blot的灰度扫描结果分析图。FIG. 6 is an analysis diagram of the grayscale scanning results of Western blot for the expression of EGFP in different treatment groups in Example 7. FIG.

图7为流式细胞仪检测结果图，其中，对照组、Kt-shEGFP+CD63-L7Ae组和Kt-shEGFP+pCDNA3.4组分别都有3个重复样。Figure 7 shows the results of flow cytometry, wherein the control group, the Kt-shEGFP+CD63-L7Ae group and the Kt-shEGFP+pCDNA3.4 group have three replicates respectively.

图8是流式细胞仪检测的柱状分析图。Figure 8 is a bar graph of flow cytometry detection.

具体实施方式Detailed ways

下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

实施例1沉默EGFP蛋白的shRNA载体构建与验证Example 1 Construction and verification of shRNA vector for silencing EGFP protein

化学合成shRNA的DNA片段Chemically synthesized DNA fragments of shRNA

(1)sh-N(无靶向性的shRNA，作为阴性对照)(SEQ ID NO:1)；(1) sh-N (shRNA without targeting, as a negative control) (SEQ ID NO: 1);

(2)Kt-sh-EGFP(带有Kt环的沉默EGFP的shRNA)(SEQ ID NO:2)；(2) Kt-sh-EGFP (shRNA with Kt loop to silence EGFP) (SEQ ID NO: 2);

以上序列由公司(Ribobio)化学合成，并构建连接到载体pRNAT-U6.1/Neo的BamHI和HindIII酶切位点之间(购自Ribobio)，分别获得得到重组质粒pRNAT-U6.1-shN和pRNAT-U6.1-Kt-shEGFP。The above sequences were chemically synthesized by the company (Ribobio), and constructed and connected between the BamHI and HindIII restriction sites of the vector pRNAT-U6.1/Neo (purchased from Ribobio), respectively, and the recombinant plasmid pRNAT-U6.1-shN was obtained. and pRNAT-U6.1-Kt-shEGFP.

实施例2表达EGFP的质粒构建Example 2 Construction of plasmid expressing EGFP

委托苏州泓迅生物科技有限公司合成EGFP基因，构建到表达载体pcDNA3.4(购自Invitrogen公司)上，获得重组质粒pcDNA3.4-EGFP，具体操作如下：Entrust Suzhou Hongxun Biotechnology Co., Ltd. to synthesize the EGFP gene, construct it on the expression vector pcDNA3.4 (purchased from Invitrogen Company), and obtain the recombinant plasmid pcDNA3.4-EGFP. The specific operations are as follows:

1.设计引物：1. Design primers:

EGFP-F:ATAAAAGGTACCATGGTGAGCAAGG；EGFP-F:ATAAAAGGTACCATGGTGAGCAAGG;

EGFP-R:ATAAAAGGATCCTTACTTGTACAGCTCG；EGFP-R:ATAAAAGGATCCTTACTTGTACAGCTCG;

2.EGFP基因扩增：2. EGFP gene amplification:

用EGFP-F和EGFP-R引物扩增EGFP基因，使扩增出来的目的基因5’端带有pCDNA3.4载体的KpnI酶切位点，3’端带有BamHI酶切位点，其中，扩增体系如下表1，扩增条件如下表2。Amplify the EGFP gene with EGFP-F and EGFP-R primers, so that the amplified target gene has the KpnI restriction site of the pCDNA3.4 vector at the 5' end and the BamHI restriction site at the 3' end, wherein, The amplification system is shown in Table 1, and the amplification conditions are shown in Table 2.

表1扩增体系：Table 1 Amplification system:

表2扩增条件Table 2 Amplification conditions

3.载体的酶切酶连3. Enzyme ligation of the vector

对目的基因和pCDNA3.4载体进行双酶切反应，目的基因双酶切反应体系如下表3，pCDNA3.4载体双酶切体系如下表4。The target gene and the pCDNA3.4 vector were subjected to a double-enzyme digestion reaction. The target gene double-enzyme digestion reaction system was shown in Table 3, and the pCDNA3.4 vector double-enzyme digestion system was shown in Table 4.

表3目的基因双酶切反应体系Table 3 Target gene double-enzyme digestion reaction system

表4 pCDNA3.4载体双酶切反应体系Table 4 pCDNA3.4 vector double digestion reaction system

具体的条件：在37℃条件下酶切30min。Specific conditions: digestion at 37°C for 30min.

双酶切后，用T4连接酶酶连，构建出重组pCDNA3.4-EGFP载体，酶连体系如下表5。After double digestion, T4 ligase was used for enzymatic ligation to construct a recombinant pCDNA3.4-EGFP vector. The enzymatic ligation system is shown in Table 5 below.

表5酶连体系：Table 5 Enzyme-linked system:

具体的条件：在16℃条件下连接过夜。Specific conditions: ligation overnight at 16°C.

4.重组质粒转化鉴定：4. Identification of recombinant plasmid transformation:

将酶连连接产物转入到E.coli DH5α(生工生物工程(上海)股份有限公司)感受态培养12小时后，挑单克隆提取质粒送到生工生物工程(上海)股份有限公司进行测序鉴定。测序结果显示正确，表明成功构建了重组pCDNA3.4-EGFP载体。The enzyme-ligated ligation product was transferred to E.coli DH5α (Sangon Bioengineering (Shanghai) Co., Ltd.) for 12 hours, and then single clone was picked to extract the plasmid and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. identification. The sequencing results were correct, indicating that the recombinant pCDNA3.4-EGFP vector was successfully constructed.

实施例3 shRNA沉默EGFP的验证Example 3 Validation of shRNA silencing EGFP

2种shRNA质粒(pRNAT-U6.1-shNP、pRNAT-U6.1-Kt-shEGFP质粒)分别与表达EGFP质粒(pcDNA3.4-EGFP)共转染293T细胞(购自生工生物工程(上海)股份有限公司)，未转染的293T细胞作为空白对照，具体操作步骤如下：Two shRNA plasmids (pRNAT-U6.1-shNP, pRNAT-U6.1-Kt-shEGFP plasmid) and EGFP expression plasmid (pcDNA3.4-EGFP) were co-transfected into 293T cells (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), and untransfected 293T cells were used as blank control. The specific operation steps are as follows:

1.转染前一天，于6孔板中每个孔铺1×10⁶个293T细胞，含10％胎牛血清(Gibco)DMEM培养基(Gibco)过夜培养。1. One day before transfection, 1×10⁶ 293T cells were plated in each well of a 6-well plate and cultured overnight in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco).

2.第二天，瞬转前把6孔板中的培养基吸取干净，加入适量无血清DMEM培养基。2. On the second day, the medium in the 6-well plate was aspirated before transient transfer, and an appropriate amount of serum-free DMEM medium was added.

3.根据每个孔需要加入总量2μg的质粒(两种质粒数量比例为1:1)，计算出所需要的质粒的量；3. Calculate the amount of plasmid required by adding a total of 2 μg of plasmid to each well (the ratio of the two plasmids is 1:1);

4.根据质粒的量，计算出所需要的转染试剂PEI(Polysciences公司，MW25000)的量，并将所需要的PEI放到60℃热水中预热；(DNA:PEI的浓度比＝1:2)；4. According to the amount of plasmid, calculate the amount of the required transfection reagent PEI (Polysciences, MW25000), and put the required PEI in 60°C hot water to preheat; (DNA:PEI concentration ratio=1: 2);

5.取灭菌的1.5mL EP管，将需要的PEI及质粒DNA分别用无血清DMEM稀释一下，然后将两者加入EP管中混合，混合液静置所需的聚合时间一般为5min左右；5. Take a sterilized 1.5mL EP tube, dilute the required PEI and plasmid DNA with serum-free DMEM respectively, then add the two to the EP tube and mix, and the polymerization time required for the mixture to stand is generally about 5min;

6.将DNA/PEI混合液加入细胞悬液中，使细胞和DNA/PEI复合物充分接触并混匀；6. Add the DNA/PEI mixture to the cell suspension to make the cells and DNA/PEI complex fully contact and mix well;

7.在37℃，5％CO₂，培养条件下进行培养，3h后补充新鲜的含10％胎牛血清的DMEM培养基至5mL，将其放到培养箱上继续培养；7. Cultivate at 37°C, 5% CO₂ , and add fresh DMEM medium containing 10% fetal bovine serum to 5 mL after 3 hours, and place it on the incubator to continue culturing;

8.瞬转后第二天检测细胞荧光，结果如图1所示，其中，各图左侧为可见光条件下的观察结果，右侧为荧光条件下的观察结果。从图中可看出，与未转染质粒的细胞(图1A)相比，转染表达质粒pcDNA3.4-EGFP和pRNAT-U6.1-sh-N质粒的细胞明显有大量荧光(图1B)，说明表达质粒可以很好地表达EGFP，shN不能沉默EGFP；而同时转染了pRNAT-U6.1-Kt-shEGFP+pcDNA3.4-EGFP的细胞，荧光大大减少(图1C)，说明这个靶向沉默EGFP的shRNA能够抑制EGFP的表达。8. Detect the cell fluorescence on the second day after transient transfer, and the results are shown in Figure 1, where the left side of each figure is the observation result under the condition of visible light, and the right side is the observation result under the fluorescence condition. As can be seen from the figure, cells transfected with plasmids expressing pcDNA3.4-EGFP and pRNAT-U6.1-sh-N showed significantly higher fluorescence compared to cells without plasmids (Fig. 1A) (Fig. 1B). ), indicating that the expression plasmid can express EGFP well, and shN cannot silence EGFP; while the cells transfected with pRNAT-U6.1-Kt-shEGFP+pcDNA3.4-EGFP, the fluorescence is greatly reduced (Fig. 1C), indicating that this shRNA targeting silencing of EGFP was able to suppress the expression of EGFP.

实施例4构建CD63-L7Ae的表达载体Example 4 Construction of the expression vector of CD63-L7Ae

1.可以与K-turn(Kt序列)相结合的蛋白1. Proteins that can bind to K-turn (Kt sequence)

搜索可以与Kt序列相结合的蛋白，再通过https://www.ncbi.nlm.nih.gov中的基因库获得相应基因序列：Search for proteins that can bind to the Kt sequence, and then obtain the corresponding gene sequence through the gene bank inhttps://www.ncbi.nlm.nih.gov :

L7Ae[Archaeoglobusfulgidus]：其氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。L7Ae[Archaeoglobusfulgidus]: its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.

NH2L1_HUMAN NHP2-like protein 1：其氨基酸序列如SEQ ID NO.5所示，核苷酸序列如SEQ ID NO.6所示。NH2L1_HUMAN NHP2-like protein 1: its amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.6.

HumanNOP56_HUMAN Nucleolar protein 56：其氨基酸序列如SEQ ID NO.7所示，核苷酸序列如SEQ ID NO.8所示。HumanNOP56_HUMAN Nucleolar protein 56: its amino acid sequence is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.8.

NOP58_HUMAN Nucleolar protein 58：其氨基酸序列如SEQ ID NO.9所示，其核苷酸序列如SEQ ID NO.10所示。NOP58_HUMAN Nucleolar protein 58: its amino acid sequence is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.10.

参考文献：references:

[1]ROZHDESTVENSKY,S.T.Binding of L7Ae protein to the K-turn ofarchaeal snoRNAs:a shared RNA binding motif for C/D and H/ACA box snoRNAs inArchaea[J].Nucleic Acids Research,31(3):869-77.[1] ROZHDESTVENSKY, S.T. Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea[J].Nucleic Acids Research,31(3):869-77 .

[2]KUHN J F,TRAN E J,STUART M E.Archaeal ribosomal protein L7 is afunctional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein[J].Nucleic Acids Research,2002,4):4.[2]KUHN J F,TRAN E J,STUART M E.Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein[J].Nucleic Acids Research,2002,4):4.

[3]OMER A D,ZIESCHE S,EBHARDT H,et al.In vitro reconstitution andactivity of a C/D box methylation guide ribonucleoprotein complex[J].Proceedings of the National Academy of Sciences of the United States ofAmerica,99(8):5289-94.[3] OMER A D, ZIESCHE S, EBHARDT H, et al. In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex [J]. Proceedings of the National Academy of Sciences of the United States of America, 99(8) :5289-94.

[4]ARNAUD,PAUL,DECEBAL,et al.Bcd1p controls RNA loading of the coreprotein Nop58 during C/D box snoRNP biogenesis[J].[4] ARNAUD, PAUL, DECEBAL, et al. Bcd1p controls RNA loading of the coreprotein Nop58 during C/D box snoRNP biogenesis[J].

2.外泌体膜蛋白的序列2. Sequences of Exosomal Membrane Proteins

外泌体膜蛋白如CD63，CD81，CD9，TP01(见中国专利申请CN201811023079.4)等蛋白都被证明可以靶向进入外泌体，其与其他蛋白融合表达，可以将目的蛋白带入外泌体，起到运载蛋白的作用。Exosome membrane proteins such as CD63, CD81, CD9, TP01 (see Chinese patent application CN201811023079.4) and other proteins have been proven to be targeted into exosomes, and their fusion expression with other proteins can bring the target protein into the exosome body, which acts as a carrier protein.

CD63：其氨基酸序列如SEQ ID NO.11所示，核苷酸序列如SEQ ID NO.12所示。CD63: Its amino acid sequence is shown in SEQ ID NO.11, and its nucleotide sequence is shown in SEQ ID NO.12.

CD81：其氨基酸序列如SEQ ID NO.13所示，核苷酸序列如SEQ ID NO.14所示。CD81: Its amino acid sequence is shown in SEQ ID NO.13, and its nucleotide sequence is shown in SEQ ID NO.14.

CD9序列：其氨基酸序列如SEQ ID NO.15所示，核苷酸序列如SEQ ID NO.16所示。CD9 sequence: its amino acid sequence is shown in SEQ ID NO.15, and its nucleotide sequence is shown in SEQ ID NO.16.

TP01序列：其氨基酸序列如SEQ ID NO.17所示，核苷酸序列如SEQ ID NO.18所示。TP01 sequence: its amino acid sequence is shown in SEQ ID NO.17, and its nucleotide sequence is shown in SEQ ID NO.18.

靶向外泌体的蛋白与Kt结合蛋白进行融合蛋白表达，将Kt结合蛋白带入外泌体，而Kt结合蛋白与Kt-shRNA上的KT环结合，又进一步将带Kt环的shRNA带入外泌体从而实现shRNA的外泌体包载。The exosome-targeting protein is expressed as a fusion protein with Kt-binding protein, which brings the Kt-binding protein into the exosome, and the Kt-binding protein binds to the KT loop on the Kt-shRNA, and further brings the shRNA with the Kt loop into the exosome. exosomes to achieve exosome encapsulation of shRNA.

3.优选以L7Ae和CD63为例，进行下列实验验证。3. It is preferable to take L7Ae and CD63 as examples to carry out the following experimental verification.

(1)设计引物F-CD63-L7Ae和R-CD63-L7Ae，引物末端引入Nhe1和BamH1的酶切位点；CD63-L7Ae基因模板由公司(生工生物工程(上海)股份有限公司)合成。两段序列由linker连接。具体构建方法参照中国专利申请CN201811023079.4实施例1。(1) The primers F-CD63-L7Ae and R-CD63-L7Ae were designed, and the restriction sites of Nhe1 and BamH1 were introduced into the ends of the primers; the CD63-L7Ae gene template was synthesized by the company (Sangon Bioengineering (Shanghai) Co., Ltd.). The two sequences are connected by a linker. For the specific construction method, refer to Example 1 of Chinese Patent Application CN201811023079.4.

相关序列信息：Related sequence information:

F-CD63-L7Ae：5’-ATAAAAGCTAGCATGGCGGTGGAAGGAGGAATGAAATGTGTG-3’F-CD63-L7Ae: 5'-ATAAAAGCTAGC ATGGCGGTGGAAGGAGGAATGAAATGTGTG-3'

R-CD63-L7Ae：5’-ATAAAAGGATCCTTAGTGGTGGTGGTGGTGGTGCTTCTGAAGG-3’R-CD63-L7Ae: 5'-ATAAAAGGATCC TTAGTGGTGGTGGTGGTGGTGCTTCTGAAGG-3'

Linker：5’-GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC-3’。Linker: 5'-GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC-3'.

实施例5含有shRNA的外泌体的制备和鉴定Example 5 Preparation and identification of exosomes containing shRNA

1.质粒共同转染293T细胞1. Co-transfection of 293T cells with plasmids

重组质粒pRNAT-U6.1-Kt-shEGFP分别与pCDNA3.4-CD63-L7Ae和空白pCDNA3.4质粒共转染293T细胞(购自生工生物工程(上海)股份有限公司)，转染方法参见实施例3。The recombinant plasmid pRNAT-U6.1-Kt-shEGFP was co-transfected with the pCDNA3.4-CD63-L7Ae and blank pCDNA3.4 plasmids respectively in 293T cells (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.). See the implementation for the transfection method. Example 3.

2.外泌体的收集和纯化2. Collection and purification of exosomes

参照中国专利申请CN201811023079.4实施例2步骤3中“外泌体的提取”的方法、步骤。Refer to the method and steps of "Extraction of Exosomes" inStep 3 of Example 2 of Chinese Patent Application CN201811023079.4.

3.外泌体的NTA检测：参照中国专利申请CN201811023079.4实施例2步骤3中“外泌体的粒径检测”的方法、步骤。NTA检测结果如图2所示，显示我们所提取的颗粒大小都在100nm左右，符合外泌体的粒径范围。3. NTA detection of exosomes: refer to the method and steps of "Exosome particle size detection" inStep 3 of Example 2 of Chinese Patent Application CN201811023079.4. The NTA detection results are shown in Figure 2, which shows that the size of the extracted particles is about 100 nm, which is in line with the particle size range of exosomes.

4.外泌体Marker蛋白和重组L7Ae蛋白的检测4. Detection of exosome Marker protein and recombinant L7Ae protein

Western blot检测外泌体的marker蛋白以及重组CD63-L7Ae蛋白量。因为L7Ae蛋白连接有His标签，采用anti-His抗体(购自proteintech，货号：66005-1-lg)来检测，具体方法参照中国专利申请CN201811023079.4实施例2步骤4“外泌体中携带重组蛋白的检测”的方法、步骤。Western blot was used to detect the marker protein of exosomes and the amount of recombinant CD63-L7Ae protein. Because the L7Ae protein is linked with a His tag, an anti-His antibody (purchased from proteintech, article number: 66005-1-lg) was used for detection. For the specific method, refer to Chinese Patent Application CN201811023079.4 Example 2,Step 4 "Exosomes carry recombinant Methods and steps for protein detection".

结果如图3所示，相比较共转染pRNAT-U6.1-Kt-shEGFP以及空白pCDNA3.4质粒的细胞所收集的外泌体(泳道1)，共转染了pRNAT-U6.1-Kt-shEGFP+pCDNA3.4CD63-L7Ae的细胞收集的外泌体(泳道2)中重组蛋白CD63和L7Ae的含量很高，说明外泌体中包载了很多CD63-L7Ae。The results are shown in Figure 3. Compared with exosomes collected from cells co-transfected with pRNAT-U6.1-Kt-shEGFP and blank pCDNA3.4 plasmid (lane 1), co-transfected with pRNAT-U6.1- The contents of recombinant proteins CD63 and L7Ae in the exosomes collected from Kt-shEGFP+pCDNA3.4CD63-L7Ae cells (lane 2) were high, indicating that a lot of CD63-L7Ae was encapsulated in the exosomes.

实施例6外泌体中shRNA的检测Example 6 Detection of shRNA in exosomes

1.外泌体RNA的提取：1. Extraction of exosomal RNA:

收集实施例5步骤2中提取的外泌体，提取外泌体总RNA，具体操作如下：Collect the exosomes extracted instep 2 of Example 5, and extract the total RNA of exosomes. The specific operations are as follows:

(1)将外泌体与1mlTrizol(Takara)混合并彻底混匀，过夜。(1) Mix the exosomes with 1ml Trizol (Takara) and mix thoroughly, overnight.

(2)带外泌体充分裂解并彻底混匀后，加入5μl 200nM的外参miDETECTTM(Ribobio)，彻底混匀，静置5-10分钟。(2) After the exosomes are fully lysed and thoroughly mixed, add 5 μl of 200nM external reference miDETECTTM (Ribobio), mix thoroughly, and let stand for 5-10 minutes.

(3)在EP管中加入400μl氯仿(购自天津百世化工有限公司)，上下剧烈震荡15秒，静置15分钟，于12000g，4℃离心15分钟。(3) Add 400 μl of chloroform (purchased from Tianjin Best Chemical Co., Ltd.) to the EP tube, shake vigorously up and down for 15 seconds, let stand for 15 minutes, and centrifuge at 12000 g at 4° C. for 15 minutes.

(4)吸取上清液360μl到新的无RNA酶的EP管中。(4) Pipette 360 μl of the supernatant into a new RNase-free EP tube.

(5)加入等体积的异丙醇(购自天津百世化工有限公司)充分混匀，静置10分钟，12000g，4℃离心15分钟。取白色沉淀，加入75％乙醇(购自天津百世化工有限公司)，小心吹打，12000g，4℃离心10分钟。(5) Add an equal volume of isopropanol (purchased from Tianjin Best Chemical Co., Ltd.) and mix well, let stand for 10 minutes, centrifuge at 12000 g for 15 minutes at 4°C. The white precipitate was taken, added with 75% ethanol (purchased from Tianjin Best Chemical Co., Ltd.), carefully pipetted, and centrifuged at 12000 g for 10 minutes at 4°C.

(6)室温晾干，加入30μl DEPC水(购自生工生物工程(上海)股份有限公司)(6) Air dry at room temperature, add 30 μl DEPC water (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.)

2.外泌体中shRNA-EGFP的检测2. Detection of shRNA-EGFP in exosomes

RT-PCR检测外泌体中shRNA的量：使用检测试剂盒(Ribobio公司，产品货号：C10211)，按照试剂盒提供的操作步骤进行检测。RT-PCR to detect the amount of shRNA in exosomes: use a detection kit (Ribobio, product number: C10211), and follow the operating steps provided by the kit.

结果如图4所示，相比较共转染pRNAT-U6.1-Kt-shEGFP以及空白pCDNA3.4质粒的细胞所收集的外泌体，共转染了pRNAT-U6.1-Kt-shEGFP+pCDNA3.4CD63-L7Ae的细胞收集的外泌体中Kt-shEGFP含量明显上升，说明外泌体膜上的L7Ae可以与Kt-shEGFP结合，并将其携带进去外泌体内。The results are shown in Figure 4. Compared with exosomes collected from cells co-transfected with pRNAT-U6.1-Kt-shEGFP and blank pCDNA3.4 plasmids, co-transfected with pRNAT-U6.1-Kt-shEGFP+ The content of Kt-shEGFP in exosomes collected from pCDNA3.4CD63-L7Ae cells was significantly increased, indicating that L7Ae on the exosome membrane can bind to Kt-shEGFP and carry it into the exosomes.

实施例7外泌体可以把所携带shRNA导入其他细胞Example 7 Exosomes can carry shRNA into other cells

将实施例5步骤2中提取得到的外泌体转染空白的293T细胞(购自生工生物工程(上海)股份有限公司)，同时转染EGFP的表达质粒pCDNA3.4-EGFP，以只转染pCDNA3.4-EGFP的293T细胞作为对照，Western blot和流式细胞仪进行观察和检测EGFP的沉默情况。The exosomes extracted instep 2 of Example 5 were transfected into blank 293T cells (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.), and the expression plasmid pCDNA3.4-EGFP of EGFP was transfected at the same time. 293T cells with pCDNA3.4-EGFP were used as control, and Western blot and flow cytometry were used to observe and detect the silencing of EGFP.

1.Western blot检测1. Western blot detection

使用EGFP抗体(购自proteintech，货号：66002-1-lg)，以及内参蛋白GAPDH的抗体(购自proteintech，货号：60004-1-lg)，具体方法参照实施例5步骤4。结果如图5、图6所示。Use EGFP antibody (purchased from proteintech, product number: 66002-1-lg), and the antibody of internal reference protein GAPDH (purchased from proteintech, product number: 60004-1-lg), the specific method refers to step 4 of Example 5. The results are shown in Figures 5 and 6.

2.流式细胞仪检测细胞中2. Flow Cytometry Detection of Cells

具体方法参照中国专利申请CN201811023079.4实施例3步骤2“流式细胞仪检测”的方法、步骤。结果如图7、图8所示。For the specific method, refer to the method and steps of "flow cytometry detection" instep 2 ofembodiment 3 of Chinese patent application CN201811023079.4. The results are shown in Figures 7 and 8.

Western blot结果显示(图5和6)和流式细胞检测的结果(图7和8)可以看出，与对照组和共转染pRNAT-U6.1-Kt-shEGFP以及空白pCDNA3.4质粒的细胞所收集的外泌体相比，含有L7Ae的外泌体组组的EGFP表达明显下降，说明Kt+CD63-L7Ae外泌体中的shEGFP能抑制EGFP的表达。Western blot results (Figures 5 and 6) and flow cytometry results (Figures 7 and 8) can be seen, compared with the control group and co-transfected pRNAT-U6.1-Kt-shEGFP and blank pCDNA3.4 plasmids Compared with exosomes collected from cells, the expression of EGFP in the exosome group containing L7Ae was significantly decreased, indicating that shEGFP in Kt+CD63-L7Ae exosomes can inhibit the expression of EGFP.

上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

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<220><220>

<223> L7Ae [Archaeoglobusfulgidus] 核苷酸序列<223> L7Ae [Archaeoglobusfulgidus] nucleotide sequence

<400> 4 <400> 4

atgtacgtga gatttgaggt tcctgaggac atgcagaacg aagctctgag tctgctggag 60atgtacgtga gatttgaggt tcctgaggac atgcagaacg aagctctgag tctgctggag 60

aaggttaggg agagcggtaa ggtaaagaaa ggtaccaacg agacgacaaa ggctgtggag 120aaggttaggg agagcggtaa ggtaaagaaa ggtaccaacg agacgacaaa ggctgtggag 120

aggggactgg caaagcttgt ttacatcgct gaggatgtcg acccaccgga aatagtggca 180aggggactgg caaagcttgt ttacatcgct gaggatgtcg acccaccgga aatagtggca 180

cacctgcccc tcctctgcga ggagaagaat gtgccgtaca tttacgttaa aagcaagaac 240cacctgcccc tcctctgcga ggagaagaat gtgccgtaca tttacgttaa aagcaagaac 240

gaccttggaa gggctgtggg cattgaggtg ccatgcgctt cggcagcgat aatcaacgag 300gaccttggaa gggctgtggg cattgaggtg ccatgcgctt cggcagcgat aatcaacgag 300

ggagagctga gaaaggagct tggaagcctt gtggagaaga ttaaaggcct tcagaagtaa 360ggagagctga gaaaggagct tggaagcctt gtggagaaga ttaaaggcct tcagaagtaa 360

<210> 5 <210> 5

<211> 128<211> 128

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> NH2L1_HUMAN NHP2-like protein 1氨基酸序列<223> NH2L1_HUMAN NHP2-like protein 1 amino acid sequence

<400> 5<400> 5

Met Thr Glu Ala Asp Val Asn Pro Lys Ala Tyr Pro Leu Ala Asp AlaMet Thr Glu Ala Asp Val Asn Pro Lys Ala Tyr Pro Leu Ala Asp Ala

1 5 10 151 5 10 15

His Leu Thr Lys Lys Leu Leu Asp Leu Val Gln Gln Ser Cys Asn TyrHis Leu Thr Lys Lys Leu Leu Asp Leu Val Gln Gln Ser Cys Asn Tyr

20 25 30 20 25 30

Lys Gln Leu Arg Lys Gly Ala Asn Glu Ala Thr Lys Thr Leu Asn ArgLys Gln Leu Arg Lys Gly Ala Asn Glu Ala Thr Lys Thr Leu Asn Arg

35 40 45 35 40 45

Gly Ile Ser Glu Phe Ile Val Met Ala Ala Asp Ala Glu Pro Leu GluGly Ile Ser Glu Phe Ile Val Met Ala Ala Asp Ala Glu Pro Leu Glu

50 55 60 50 55 60

Ile Ile Leu His Leu Pro Leu Leu Cys Glu Asp Lys Asn Val Pro TyrIle Ile Leu His Leu Pro Leu Leu Cys Glu Asp Lys Asn Val Pro Tyr

65 70 75 8065 70 75 80

Val Phe Val Arg Ser Lys Gln Ala Leu Gly Arg Ala Cys Gly Val SerVal Phe Val Arg Ser Lys Gln Ala Leu Gly Arg Ala Cys Gly Val Ser

85 90 95 85 90 95

Arg Pro Val Ile Ala Cys Ser Val Thr Ile Lys Glu Gly Ser Gln LeuArg Pro Val Ile Ala Cys Ser Val Thr Ile Lys Glu Gly Ser Gln Leu

100 105 110 100 105 110

Lys Gln Gln Ile Gln Ser Ile Gln Gln Ser Ile Glu Arg Leu Leu ValLys Gln Gln Ile Gln Ser Ile Gln Gln Ser Ile Glu Arg Leu Leu Val

115 120 125 115 120 125

<210> 6<210> 6

<211> 387<211> 387

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> NH2L1_HUMAN NHP2-like protein 1核苷酸序列<223> NH2L1_HUMAN NHP2-like protein 1 nucleotide sequence

<400> 6<400> 6

atgactgagg ctgatgtgaa tccaaaggcc tatccccttg ccgatgccca cctcaccaag 60atgactgagg ctgatgtgaa tccaaaggcc tatccccttg ccgatgccca cctcaccaag 60

aagctactgg acctcgttca gcagtcatgt aactataagc agcttcggaa aggagccaat 120aagctactgg acctcgttca gcagtcatgt aactataagc agcttcggaa aggagccaat 120

gaggccacca aaaccctcaa caggggcatc tctgagttca tcgtgatggc tgcagacgcc 180gaggccacca aaaccctcaa caggggcatc tctgagttca tcgtgatggc tgcagacgcc 180

gagccactgg agatcattct gcacctgccg ctgctgtgtg aagacaagaa tgtgccctac 240gagccactgg agatcattct gcacctgccg ctgctgtgtg aagacaagaa tgtgccctac 240

gtgtttgtgc gctccaagca ggccctgggg agagcctgtg gggtctccag gcctgtcatc 300gtgtttgtgc gctccaagca ggccctgggg agagcctgtg gggtctccag gcctgtcatc 300

gcctgttctg tcaccatcaa agaaggctcg cagctgaaac agcagatcca atccattcag 360gcctgttctg tcaccatcaa agaaggctcg cagctgaaac agcagatcca atccattcag 360

cagtccattg aaaggctctt agtctaa 387cagtccattg aaaggctctt agtctaa 387

<210> 7<210> 7

<211> 594<211> 594

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> HumanNOP56_HUMAN Nucleolar protein 56氨基酸序列<223> HumanNOP56_HUMAN Nucleolar protein 56 amino acid sequence

<400> 7 <400> 7

Met Val Leu Leu His Val Leu Phe Glu His Ala Val Gly Tyr Ala LeuMet Val Leu Leu His Val Leu Phe Glu His Ala Val Gly Tyr Ala Leu

1 5 10 151 5 10 15

Leu Ala Leu Lys Glu Val Glu Glu Ile Ser Leu Leu Gln Pro Gln ValLeu Ala Leu Lys Glu Val Glu Glu Ile Ser Leu Leu Gln Pro Gln Val

20 25 30 20 25 30

Glu Glu Ser Val Leu Asn Leu Gly Lys Phe His Ser Ile Val Arg LeuGlu Glu Ser Val Leu Asn Leu Gly Lys Phe His Ser Ile Val Arg Leu

35 40 45 35 40 45

Val Ala Phe Cys Pro Phe Ala Ser Ser Gln Val Ala Leu Glu Asn AlaVal Ala Phe Cys Pro Phe Ala Ser Ser Gln Val Ala Leu Glu Asn Ala

50 55 60 50 55 60

Asn Ala Val Ser Glu Gly Val Val His Glu Asp Leu Arg Leu Leu LeuAsn Ala Val Ser Glu Gly Val Val His Glu Asp Leu Arg Leu Leu Leu

65 70 75 8065 70 75 80

Glu Thr His Leu Pro Ser Lys Lys Lys Lys Val Leu Leu Gly Val GlyGlu Thr His Leu Pro Ser Lys Lys Lys Lys Lys Val Leu Leu Gly Val Gly

85 90 95 85 90 95

Asp Pro Lys Ile Gly Ala Ala Ile Gln Glu Glu Leu Gly Tyr Asn CysAsp Pro Lys Ile Gly Ala Ala Ile Gln Glu Glu Leu Gly Tyr Asn Cys

100 105 110 100 105 110

Gln Thr Gly Gly Val Ile Ala Glu Ile Leu Arg Gly Val Arg Leu HisGln Thr Gly Gly Val Ile Ala Glu Ile Leu Arg Gly Val Arg Leu His

115 120 125 115 120 125

Phe His Asn Leu Val Lys Gly Leu Thr Asp Leu Ser Ala Cys Lys AlaPhe His Asn Leu Val Lys Gly Leu Thr Asp Leu Ser Ala Cys Lys Ala

130 135 140 130 135 140

Gln Leu Gly Leu Gly His Ser Tyr Ser Arg Ala Lys Val Lys Phe AsnGln Leu Gly Leu Gly His Ser Tyr Ser Arg Ala Lys Val Lys Phe Asn

145 150 155 160145 150 155 160

Val Asn Arg Val Asp Asn Met Ile Ile Gln Ser Ile Ser Leu Leu AspVal Asn Arg Val Asp Asn Met Ile Ile Gln Ser Ile Ser Leu Leu Asp

165 170 175 165 170 175

Gln Leu Asp Lys Asp Ile Asn Thr Phe Ser Met Arg Val Arg Glu TrpGln Leu Asp Lys Asp Ile Asn Thr Phe Ser Met Arg Val Arg Glu Trp

180 185 190 180 185 190

Tyr Gly Tyr His Phe Pro Glu Leu Val Lys Ile Ile Asn Asp Asn AlaTyr Gly Tyr His Phe Pro Glu Leu Val Lys Ile Ile Asn Asp Asn Ala

195 200 205 195 200 205

Thr Tyr Cys Arg Leu Ala Gln Phe Ile Gly Asn Arg Arg Glu Leu AsnThr Tyr Cys Arg Leu Ala Gln Phe Ile Gly Asn Arg Arg Glu Leu Asn

210 215 220 210 215 220

Glu Asp Lys Leu Glu Lys Leu Glu Glu Leu Thr Met Asp Gly Ala LysGlu Asp Lys Leu Glu Lys Leu Glu Glu Leu Thr Met Asp Gly Ala Lys

225 230 235 240225 230 235 240

Ala Lys Ala Ile Leu Asp Ala Ser Arg Ser Ser Met Gly Met Asp IleAla Lys Ala Ile Leu Asp Ala Ser Arg Ser Ser Met Gly Met Asp Ile

245 250 255 245 250 255

Ser Ala Ile Asp Leu Ile Asn Ile Glu Ser Phe Ser Ser Arg Val ValSer Ala Ile Asp Leu Ile Asn Ile Glu Ser Phe Ser Ser Arg Val Val

260 265 270 260 265 270

Ser Leu Ser Glu Tyr Arg Gln Ser Leu His Thr Tyr Leu Arg Ser LysSer Leu Ser Glu Tyr Arg Gln Ser Leu His Thr Tyr Leu Arg Ser Lys

275 280 285 275 280 285

Met Ser Gln Val Ala Pro Ser Leu Ser Ala Leu Ile Gly Glu Ala ValMet Ser Gln Val Ala Pro Ser Leu Ser Ala Leu Ile Gly Glu Ala Val

290 295 300 290 295 300

Gly Ala Arg Leu Ile Ala His Ala Gly Ser Leu Thr Asn Leu Ala LysGly Ala Arg Leu Ile Ala His Ala Gly Ser Leu Thr Asn Leu Ala Lys

305 310 315 320305 310 315 320

Tyr Pro Ala Ser Thr Val Gln Ile Leu Gly Ala Glu Lys Ala Leu PheTyr Pro Ala Ser Thr Val Gln Ile Leu Gly Ala Glu Lys Ala Leu Phe

325 330 335 325 330 335

Arg Ala Leu Lys Thr Arg Gly Asn Thr Pro Lys Tyr Gly Leu Ile PheArg Ala Leu Lys Thr Arg Gly Asn Thr Pro Lys Tyr Gly Leu Ile Phe

340 345 350 340 345 350

His Ser Thr Phe Ile Gly Arg Ala Ala Ala Lys Asn Lys Gly Arg IleHis Ser Thr Phe Ile Gly Arg Ala Ala Ala Lys Asn Lys Gly Arg Ile

355 360 365 355 360 365

Ser Arg Tyr Leu Ala Asn Lys Cys Ser Ile Ala Ser Arg Ile Asp CysSer Arg Tyr Leu Ala Asn Lys Cys Ser Ile Ala Ser Arg Ile Asp Cys

370 375 380 370 375 380

Phe Ser Glu Val Pro Thr Ser Val Phe Gly Glu Lys Leu Arg Glu GlnPhe Ser Glu Val Pro Thr Ser Val Phe Gly Glu Lys Leu Arg Glu Gln

385 390 395 400385 390 395 400

Val Glu Glu Arg Leu Ser Phe Tyr Glu Thr Gly Glu Ile Pro Arg LysVal Glu Glu Arg Leu Ser Phe Tyr Glu Thr Gly Glu Ile Pro Arg Lys

405 410 415 405 410 415

Asn Leu Asp Val Met Lys Glu Ala Met Val Gln Ala Glu Glu Ala AlaAsn Leu Asp Val Met Lys Glu Ala Met Val Gln Ala Glu Glu Ala Ala

420 425 430 420 425 430

Ala Glu Ile Thr Arg Lys Leu Glu Lys Gln Glu Lys Lys Arg Leu LysAla Glu Ile Thr Arg Lys Leu Glu Lys Gln Glu Lys Lys Arg Leu Lys

435 440 445 435 440 445

Lys Glu Lys Lys Arg Leu Ala Ala Leu Ala Leu Ala Ser Ser Glu AsnLys Glu Lys Lys Arg Leu Ala Ala Leu Ala Leu Ala Ser Ser Glu Asn

450 455 460 450 455 460

Ser Ser Ser Thr Pro Glu Glu Cys Glu Glu Met Ser Glu Lys Pro LysSer Ser Ser Thr Pro Glu Glu Cys Glu Glu Met Ser Glu Lys Pro Lys

465 470 475 480465 470 475 480

Lys Lys Lys Lys Gln Lys Pro Gln Glu Val Pro Gln Glu Asn Gly MetLys Lys Lys Lys Gln Lys Pro Gln Glu Val Pro Gln Glu Asn Gly Met

485 490 495 485 490 495

Glu Asp Pro Ser Ile Ser Phe Ser Lys Pro Lys Lys Lys Lys Ser PheGlu Asp Pro Ser Ile Ser Phe Ser Lys Pro Lys Lys Lys Lys Ser Phe

500 505 510 500 505 510

Ser Lys Glu Glu Leu Met Ser Ser Asp Leu Glu Glu Thr Ala Gly SerSer Lys Glu Glu Leu Met Ser Ser Asp Leu Glu Glu Thr Ala Gly Ser

515 520 525 515 520 525

Thr Ser Ile Pro Lys Arg Lys Lys Ser Thr Pro Lys Glu Glu Thr ValThr Ser Ile Pro Lys Arg Lys Lys Ser Thr Pro Lys Glu Glu Thr Val

530 535 540 530 535 540

Asn Asp Pro Glu Glu Ala Gly His Arg Ser Gly Ser Lys Lys Lys ArgAsn Asp Pro Glu Glu Ala Gly His Arg Ser Gly Ser Lys Lys Lys Arg

545 550 555 560545 550 555 560

Lys Phe Ser Lys Glu Glu Pro Val Ser Ser Gly Pro Glu Glu Ala ValLys Phe Ser Lys Glu Glu Pro Val Ser Ser Gly Pro Glu Glu Ala Val

565 570 575 565 570 575

Gly Lys Ser Ser Ser Lys Lys Lys Lys Lys Phe His Lys Ala Ser GlnGly Lys Ser Ser Ser Lys Lys Lys Lys Lys Lys Phe His Lys Ala Ser Gln

580 585 590 580 585 590

Glu AspGlu Asp

<210> 8<210> 8

<211> 1785<211> 1785

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220> <220>

<223> HumanNOP56_HUMAN Nucleolar protein 56核苷酸序列<223> HumanNOP56_HUMAN Nucleolar protein 56 nucleotide sequence

<400> 8<400> 8

atggtgctgt tgcacgtgct gtttgagcac gcggtcggct acgcgctgct ggcgctgaag 60atggtgctgt tgcacgtgct gtttgagcac gcggtcggct acgcgctgct ggcgctgaag 60

gaagtggagg agatcagtct gctgcagccg caggtggagg agtctgtgct caacctgggc 120gaagtggagg agatcagtct gctgcagccg caggtggagg agtctgtgct caacctgggc 120

aaattccaca gcatcgttcg tctggtggcc ttttgtccct ttgcctcatc ccaggttgcc 180aaattccaca gcatcgttcg tctggtggcc ttttgtccct ttgcctcatc ccaggttgcc 180

ttggaaaatg ccaacgccgt gtctgaaggg gttgttcatg aggacctccg cctgctcttg 240ttggaaaatg ccaacgccgt gtctgaaggg gttgttcatg aggacctccg cctgctcttg 240

gagacccacc tgccgtccaa aaagaagaaa gtactcttgg gagttgggga tcccaagatt 300gagacccacc tgccgtccaa aaagaagaaa gtactcttgg gagttgggga tcccaagatt 300

ggtgccgcaa tacaggagga gttagggtac aactgccaga ctggaggagt catagctgag 360ggtgccgcaa tacaggagga gttagggtac aactgccaga ctggaggagt catagctgag 360

atcctgcgag gagttcgtct gcacttccac aatctggtga agggtctgac cgatctgtca 420atcctgcgag gagttcgtct gcacttccac aatctggtga agggtctgac cgatctgtca 420

gcttgtaaag cacagctggg gctgggacac agctattccc gtgccaaagt taagtttaat 480gcttgtaaag cacagctggg gctgggacac agctattccc gtgccaaagt taagtttaat 480

gtgaaccggg tggacaatat gatcatccag tccattagcc tcctggacca gctggataag 540gtgaaccggg tggacaatat gatcatccag tccattagcc tcctggacca gctggataag 540

gacatcaata ccttctctat gcgtgtcagg gagtggtacg ggtatcactt tccggagctg 600gacatcaata ccttctctat gcgtgtcagg gagtggtacg ggtatcactt tccggagctg 600

gtgaagatca tcaacgacaa tgccacatac tgccgtcttg cccagtttat tggaaaccga 660gtgaagatca tcaacgacaa tgccacatac tgccgtcttg cccagtttat tggaaaccga 660

agggaactga atgaggacaa gctggagaag ctggaggagc tgacaatgga tggggccaag 720agggaactga atgaggacaa gctggagaag ctggaggagc tgacaatgga tggggccaag 720

gctaaggcta ttctggatgc ctcacggtcc tccatgggca tggacatatc tgccattgac 780gctaaggcta ttctggatgc ctcacggtcc tccatgggca tggacatatc tgccattgac 780

ttgataaaca tcgagagctt ctccagtcgt gtggtgtctt tatctgaata ccgccagagc 840ttgataaaca tcgagagctt ctccagtcgt gtggtgtctt tatctgaata ccgccagagc 840

ctacacactt acctgcgctc caagatgagc caagtagccc ccagcctgtc agccctaatt 900ctacacactt acctgcgctc caagatgagc caagtagccc ccagcctgtc agccctaatt 900

ggggaagcgg taggtgcacg tctcatcgca catgctggca gcctcaccaa cctggccaag 960ggggaagcgg taggtgcacg tctcatcgca catgctggca gcctcaccaa cctggccaag 960

tatccagcat ccacagtgca gatccttggg gctgaaaagg ccctgttcag agccctgaag 1020tatccagcat ccacagtgca gatccttggg gctgaaaagg ccctgttcag agccctgaag 1020

acaaggggta acactccaaa atatggactc attttccact ccaccttcat tggccgagca 1080acaaggggta acactccaaa atatggactc attttccact ccaccttcat tggccgagca 1080

gctgccaaga acaaaggccg catctcccga tacctggcaa acaaatgcag tattgcctca 1140gctgccaaga acaaaggccg catctcccga tacctggcaa acaaatgcag tattgcctca 1140

cgaatcgatt gcttctctga ggtgcccacg agtgtattcg gggagaagct tcgagaacaa 1200cgaatcgatt gcttctctga ggtgcccacg agtgtattcg gggagaagct tcgagaacaa 1200

gttgaagagc gactgtcctt ctatgagact ggagagatac cacgaaagaa tctggatgtc 1260gttgaagagc gactgtcctt ctatgagact ggagagatac cacgaaagaa tctggatgtc 1260

atgaaggaag caatggttca ggcagaggaa gcggctgctg agattactag gaagctggag 1320atgaaggaag caatggttca ggcagaggaa gcggctgctg agattactag gaagctggag 1320

aaacaggaga agaaacgctt aaagaaggaa aagaaacggc tggctgcact tgccctcgcg 1380aaacaggaga agaaacgctt aaagaaggaa aagaaacggc tggctgcact tgccctcgcg 1380

tcttcagaaa acagcagtag tactccagag gagtgtgagg agatgagtga aaaacccaaa 1440tcttcagaaa acagcagtag tactccagag gagtgtgagg agatgagtga aaaacccaaa 1440

aagaagaaaa agcaaaagcc ccaggaggtt cctcaggaga atggaatgga agacccatct 1500aagaagaaaa agcaaaagcc ccaggaggtt cctcaggaga atggaatgga agacccatct 1500

atctctttct ccaaacccaa gaaaaagaaa tctttttcca aggaggagtt gatgagtagc 1560atctctttct ccaaacccaa gaaaaagaaa tctttttcca aggaggagtt gatgagtagc 1560

gatcttgaag agaccgctgg cagcaccagt attcccaaga ggaagaagtc tacacccaag 1620gatcttgaag agaccgctgg cagcaccagt attcccaaga ggaagaagtc tacacccaag 1620

gaggaaacag ttaatgaccc tgaggaggca ggccacagaa gtggctccaa gaaaaagagg 1680gaggaaacag ttaatgaccc tgaggaggca ggccacagaa gtggctccaa gaaaaagagg 1680

aaattctcca aagaggagcc ggtcagcagt gggcctgaag aggcggttgg caagagcagc 1740aaattctcca aagaggagcc ggtcagcagt gggcctgaag aggcggttgg caagagcagc 1740

tccaagaaga agaaaaagtt ccataaagca tcccaggaag attag 1785tccaagaaga agaaaaagtt ccataaagca tcccaggaag attag 1785

<210> 9 <210> 9

<211> 529<211> 529

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> HumanNOP58_HUMAN Nucleolar protein 58氨基酸序列<223> HumanNOP58_HUMAN Nucleolar protein 58 amino acid sequence

<400> 9 <400> 9

Met Leu Val Leu Phe Glu Thr Ser Val Gly Tyr Ala Ile Phe Lys ValMet Leu Val Leu Phe Glu Thr Ser Val Gly Tyr Ala Ile Phe Lys Val

1 5 10 151 5 10 15

Leu Asn Glu Lys Lys Leu Gln Glu Val Asp Ser Leu Trp Lys Glu PheLeu Asn Glu Lys Lys Leu Gln Glu Val Asp Ser Leu Trp Lys Glu Phe

20 25 30 20 25 30

Glu Thr Pro Glu Lys Ala Asn Lys Ile Val Lys Leu Lys His Phe GluGlu Thr Pro Glu Lys Ala Asn Lys Ile Val Lys Leu Lys His Phe Glu

35 40 45 35 40 45

Lys Phe Gln Asp Thr Ala Glu Ala Leu Ala Ala Phe Thr Ala Leu MetLys Phe Gln Asp Thr Ala Glu Ala Leu Ala Ala Phe Thr Ala Leu Met

50 55 60 50 55 60

Glu Gly Lys Ile Asn Lys Gln Leu Lys Lys Val Leu Lys Lys Ile ValGlu Gly Lys Ile Asn Lys Gln Leu Lys Lys Val Leu Lys Lys Ile Val

65 70 75 8065 70 75 80

Lys Glu Ala His Glu Pro Leu Ala Val Ala Asp Ala Lys Leu Gly GlyLys Glu Ala His Glu Pro Leu Ala Val Ala Asp Ala Lys Leu Gly Gly

85 90 95 85 90 95

Val Ile Lys Glu Lys Leu Asn Leu Ser Cys Ile His Ser Pro Val ValVal Ile Lys Glu Lys Leu Asn Leu Ser Cys Ile His Ser Pro Val Val

100 105 110 100 105 110

Asn Glu Leu Met Arg Gly Ile Arg Ser Gln Met Asp Gly Leu Ile ProAsn Glu Leu Met Arg Gly Ile Arg Ser Gln Met Asp Gly Leu Ile Pro

115 120 125 115 120 125

Gly Val Glu Pro Arg Glu Met Ala Ala Met Cys Leu Gly Leu Ala HisGly Val Glu Pro Arg Glu Met Ala Ala Met Cys Leu Gly Leu Ala His

130 135 140 130 135 140

Ser Leu Ser Arg Tyr Arg Leu Lys Phe Ser Ala Asp Lys Val Asp ThrSer Leu Ser Arg Tyr Arg Leu Lys Phe Ser Ala Asp Lys Val Asp Thr

145 150 155 160145 150 155 160

Met Ile Val Gln Ala Ile Ser Leu Leu Asp Asp Leu Asp Lys Glu LeuMet Ile Val Gln Ala Ile Ser Leu Leu Asp Asp Leu Asp Lys Glu Leu

165 170 175 165 170 175

Asn Asn Tyr Ile Met Arg Cys Arg Glu Trp Tyr Gly Trp His Phe ProAsn Asn Tyr Ile Met Arg Cys Arg Glu Trp Tyr Gly Trp His Phe Pro

180 185 190 180 185 190

Glu Leu Gly Lys Ile Ile Ser Asp Asn Leu Thr Tyr Cys Lys Cys LeuGlu Leu Gly Lys Ile Ile Ser Asp Asn Leu Thr Tyr Cys Lys Cys Leu

195 200 205 195 200 205

Gln Lys Val Gly Asp Arg Lys Asn Tyr Ala Ser Ala Lys Leu Ser GluGln Lys Val Gly Asp Arg Lys Asn Tyr Ala Ser Ala Lys Leu Ser Glu

210 215 220 210 215 220

Leu Leu Pro Glu Glu Val Glu Ala Glu Val Lys Ala Ala Ala Glu IleLeu Leu Pro Glu Glu Val Glu Ala Glu Val Lys Ala Ala Ala Glu Ile

225 230 235 240225 230 235 240

Ser Met Gly Thr Glu Val Ser Glu Glu Asp Ile Cys Asn Ile Leu HisSer Met Gly Thr Glu Val Ser Glu Glu Asp Ile Cys Asn Ile Leu His

245 250 255 245 250 255

Leu Cys Thr Gln Val Ile Glu Ile Ser Glu Tyr Arg Thr Gln Leu TyrLeu Cys Thr Gln Val Ile Glu Ile Ser Glu Tyr Arg Thr Gln Leu Tyr

260 265 270 260 265 270

Glu Tyr Leu Gln Asn Arg Met Met Ala Ile Ala Pro Asn Val Thr ValGlu Tyr Leu Gln Asn Arg Met Met Ala Ile Ala Pro Asn Val Thr Val

275 280 285 275 280 285

Met Val Gly Glu Leu Val Gly Ala Arg Leu Ile Ala His Ala Gly SerMet Val Gly Glu Leu Val Gly Ala Arg Leu Ile Ala His Ala Gly Ser

290 295 300 290 295 300

Leu Leu Asn Leu Ala Lys His Ala Ala Ser Thr Val Gln Ile Leu GlyLeu Leu Asn Leu Ala Lys His Ala Ala Ser Thr Val Gln Ile Leu Gly

305 310 315 320305 310 315 320

Ala Glu Lys Ala Leu Phe Arg Ala Leu Lys Ser Arg Arg Asp Thr ProAla Glu Lys Ala Leu Phe Arg Ala Leu Lys Ser Arg Arg Asp Thr Pro

325 330 335 325 330 335

Lys Tyr Gly Leu Ile Tyr His Ala Ser Leu Val Gly Gln Thr Ser ProLys Tyr Gly Leu Ile Tyr His Ala Ser Leu Val Gly Gln Thr Ser Pro

340 345 350 340 345 350

Lys His Lys Gly Lys Ile Ser Arg Met Leu Ala Ala Lys Thr Val LeuLys His Lys Gly Lys Ile Ser Arg Met Leu Ala Ala Lys Thr Val Leu

355 360 365 355 360 365

Ala Ile Arg Tyr Asp Ala Phe Gly Glu Asp Ser Ser Ser Ala Met GlyAla Ile Arg Tyr Asp Ala Phe Gly Glu Asp Ser Ser Ser Ala Met Gly

370 375 380 370 375 380

Val Glu Asn Arg Ala Lys Leu Glu Ala Arg Leu Arg Thr Leu Glu AspVal Glu Asn Arg Ala Lys Leu Glu Ala Arg Leu Arg Thr Leu Glu Asp

385 390 395 400385 390 395 400

Arg Gly Ile Arg Lys Ile Ser Gly Thr Gly Lys Ala Leu Ala Lys ThrArg Gly Ile Arg Lys Ile Ser Gly Thr Gly Lys Ala Leu Ala Lys Thr

405 410 415 405 410 415

Glu Lys Tyr Glu His Lys Ser Glu Val Lys Thr Tyr Asp Pro Ser GlyGlu Lys Tyr Glu His Lys Ser Glu Val Lys Thr Tyr Asp Pro Ser Gly

420 425 430 420 425 430

Asp Ser Thr Leu Pro Thr Cys Ser Lys Lys Arg Lys Ile Glu Gln ValAsp Ser Thr Leu Pro Thr Cys Ser Lys Lys Arg Lys Ile Glu Gln Val

435 440 445 435 440 445

Asp Lys Glu Asp Glu Ile Thr Glu Lys Lys Ala Lys Lys Ala Lys IleAsp Lys Glu Asp Glu Ile Thr Glu Lys Lys Ala Lys Lys Ala Lys Ile

450 455 460 450 455 460

Lys Val Lys Val Glu Glu Glu Glu Glu Glu Lys Val Ala Glu Glu GluLys Val Lys Val Glu Glu Glu Glu Glu Glu Glu Lys Val Ala Glu Glu Glu

465 470 475 480465 470 475 480

Glu Thr Ser Val Lys Lys Lys Lys Lys Arg Gly Lys Lys Lys His IleGlu Thr Ser Val Lys Lys Lys Lys Lys Lys Arg Gly Lys Lys Lys His His Ile

485 490 495 485 490 495

Lys Glu Glu Pro Leu Ser Glu Glu Glu Pro Cys Thr Ser Thr Ala IleLys Glu Glu Pro Leu Ser Glu Glu Glu Pro Cys Thr Ser Thr Ala Ile

500 505 510 500 505 510

Ala Ser Pro Glu Lys Lys Lys Lys Lys Lys Lys Lys Arg Glu Asn GluAla Ser Pro Glu Lys Lys Lys Lys Lys Lys Lys Lys Lys Arg Glu Asn Glu

515 520 525 515 520 525

AspAsp

<210> 10<210> 10

<211> 1590<211> 1590

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> HumanNOP58_HUMAN Nucleolar protein 58核苷酸序列<223> HumanNOP58_HUMAN Nucleolar protein 58 nucleotide sequence

<400> 10<400> 10

atgttggtgc tgtttgaaac gtctgtgggt tacgccatct ttaaggttct aaatgagaag 60atgttggtgc tgtttgaaac gtctgtgggt tacgccatct ttaaggttct aaatgagaag 60

aaacttcaag aggttgatag tttatggaaa gaatttgaaa ctccagagaa agcaaacaaa 120aaacttcaag aggttgatag tttatggaaa gaatttgaaa ctccagagaa agcaaacaaa 120

atagtaaagc taaaacattt tgagaaattt caggatacag cagaagcatt agcagcattc 180atagtaaagc taaaacattt tgagaaattt caggatacag cagaagcatt agcagcattc 180

acagctctga tggagggcaa aatcaataag cagctgaaaa aagttctgaa gaaaatagta 240acagctctga tggagggcaa aatcaataag cagctgaaaa aagttctgaa gaaaatagta 240

aaagaagccc atgaaccgct ggcagtagct gatgctaaac taggaggggt cataaaggaa 300aaagaagccc atgaaccgct ggcagtagct gatgctaaac taggaggggt cataaaggaa 300

aagctgaatc tcagttgtat ccatagtcct gttgttaatg aacttatgag aggaattcgt 360aagctgaatc tcagttgtat ccatagtcct gttgttaatg aacttatgag aggaattcgt 360

tcacaaatgg atggattaat ccctggggta gaaccacgtg aaatggcagc tatgtgtctt 420tcacaaatgg atggattaat ccctggggta gaaccacgtg aaatggcagc tatgtgtctt 420

ggattggctc acagcctgtc tcgatataga ttgaagttta gcgctgataa agtagacaca 480ggattggctc acagcctgtc tcgatataga ttgaagttta gcgctgataa agtagacaca 480

atgattgttc aggcaatttc cttgttagat gacttggata aagaactaaa caactacatt 540atgattgttc aggcaatttc cttgttagat gacttggata aagaactaaa caactacatt 540

atgcgatgta gagaatggta tggctggcat ttccctgaat taggaaaaat tatttcagat 600atgcgatgta gagaatggta tggctggcat ttccctgaat taggaaaaat tatttcagat 600

aatttaacat actgcaagtg tttacagaaa gttggcgata ggaagaacta tgcctctgcc 660aatttaacat actgcaagtg tttacagaaa gttggcgata ggaagaacta tgcctctgcc 660

aagctttctg agttgctgcc agaagaagtt gaagcagaag tgaaagcagc tgcagagata 720aagctttctg agttgctgcc agaagaagtt gaagcagaag tgaaagcagc tgcagagata 720

tcaatgggaa cagaggtttc agaagaagat atttgcaata ttctgcatct ttgcacccag 780tcaatgggaa cagaggtttc agaagaagat atttgcaata ttctgcatct ttgcacccag 780

gtgattgaaa tctctgaata tcgaacccag ctctatgaat atctacaaaa tcgaatgatg 840gtgattgaaa tctctgaata tcgaacccag ctctatgaat atctacaaaa tcgaatgatg 840

gccattgcac ccaatgttac agtcatggtt ggggaattag ttggagcacg gcttattgct 900gccattgcac ccaatgttac agtcatggtt ggggaattag ttggagcacg gcttattgct 900

catgcaggtt ctcttttaaa tttggccaag catgcagctt ctaccgttca gattcttgga 960catgcaggtt ctcttttaaa tttggccaag catgcagctt ctaccgttca gattcttgga 960

gctgaaaagg cacttttcag agccctcaaa tctagacggg atacccctaa gtatggtctc 1020gctgaaaagg cacttttcag agccctcaaa tctagacggg atacccctaa gtatggtctc 1020

atttatcatg cttcactcgt gggccagaca agtcccaaac acaaaggaaa gatttctcga 1080atttatcatg cttcactcgt gggccagaca agtcccaaac acaaaggaaa gatttctcga 1080

atgctggcag ccaaaaccgt tttggctatc cgttatgatg cttttggtga ggattcaagt 1140atgctggcag ccaaaaccgt tttggctatc cgttatgatg cttttggtga ggattcaagt 1140

tctgcaatgg gagttgagaa cagagccaaa ttagaggcca ggttgagaac tttggaagac 1200tctgcaatgg gagttgagaa cagagccaaa ttagaggcca ggttgagaac tttggaagac 1200

agagggataa gaaaaataag tggaacagga aaagcattag caaaaacaga aaaatatgaa 1260agagggataa gaaaaataag tggaacagga aaagcattag caaaaacaga aaaatatgaa 1260

cacaaaagtg aagtgaagac ttacgatcct tctggtgact ccacacttcc aacctgttct 1320cacaaaagtg aagtgaagac ttacgatcct tctggtgact ccacacttcc aacctgttct 1320

aaaaaacgca aaatagaaca ggtagataaa gaggatgaaa ttactgaaaa gaaagccaaa 1380aaaaaacgca aaatagaaca ggtagataaa gaggatgaaa ttactgaaaa gaaagccaaa 1380

aaagccaaga ttaaagttaa agttgaagaa gaggaagaag aaaaagtggc agaagaagaa 1440aaagccaaga ttaaagttaa agttgaagaa gaggaagaag aaaaagtggc agaagaagaa 1440

gaaacatctg tgaagaagaa gaagaaaagg ggtaaaaaga aacacattaa ggaagaacca 1500gaaacatctg tgaagaagaa gaagaaaagg ggtaaaaaga aacacattaa ggaagaacca 1500

ctttctgagg aagaaccatg taccagcaca gcaattgcta gtccagagaa aaagaagaaa 1560ctttctgagg aagaaccatg taccagcaca gcaattgcta gtccagagaa aaagaagaaa 1560

aagaaaaaaa agagagagaa cgaggattaa 1590aagaaaaaaa agagagagaa cgaggattaa 1590

<210> 11<210> 11

<211> 238<211> 238

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> CD63氨基酸序列<223> CD63 amino acid sequence

<400> 11<400> 11

Met Ala Val Glu Gly Gly Met Lys Cys Val Lys Phe Leu Leu Tyr ValMet Ala Val Glu Gly Gly Met Lys Cys Val Lys Phe Leu Leu Tyr Val

1 5 10 151 5 10 15

Leu Leu Leu Ala Phe Cys Ala Cys Ala Val Gly Leu Ile Ala Val GlyLeu Leu Leu Ala Phe Cys Ala Cys Ala Val Gly Leu Ile Ala Val Gly

20 25 30 20 25 30

Val Gly Ala Gln Leu Val Leu Ser Gln Thr Ile Ile Gln Gly Ala ThrVal Gly Ala Gln Leu Val Leu Ser Gln Thr Ile Ile Gln Gly Ala Thr

35 40 45 35 40 45

Pro Gly Ser Leu Leu Pro Val Val Ile Ile Ala Val Gly Val Phe LeuPro Gly Ser Leu Leu Pro Val Val Ile Ile Ala Val Gly Val Phe Leu

50 55 60 50 55 60

Phe Leu Val Ala Phe Val Gly Cys Cys Gly Ala Cys Lys Glu Asn TyrPhe Leu Val Ala Phe Val Gly Cys Cys Gly Ala Cys Lys Glu Asn Tyr

65 70 75 8065 70 75 80

Cys Leu Met Ile Thr Phe Ala Ile Phe Leu Ser Leu Ile Met Leu ValCys Leu Met Ile Thr Phe Ala Ile Phe Leu Ser Leu Ile Met Leu Val

85 90 95 85 90 95

Glu Val Ala Ala Ala Ile Ala Gly Tyr Val Phe Arg Asp Lys Val MetGlu Val Ala Ala Ala Ile Ala Gly Tyr Val Phe Arg Asp Lys Val Met

100 105 110 100 105 110

Ser Glu Phe Asn Asn Asn Phe Arg Gln Gln Met Glu Asn Tyr Pro LysSer Glu Phe Asn Asn Asn Phe Arg Gln Gln Met Glu Asn Tyr Pro Lys

115 120 125 115 120 125

Asn Asn His Thr Ala Ser Ile Leu Asp Arg Met Gln Ala Asp Phe LysAsn Asn His Thr Ala Ser Ile Leu Asp Arg Met Gln Ala Asp Phe Lys

130 135 140 130 135 140

Cys Cys Gly Ala Ala Asn Tyr Thr Asp Trp Glu Lys Ile Pro Ser MetCys Cys Gly Ala Ala Asn Tyr Thr Asp Trp Glu Lys Ile Pro Ser Met

145 150 155 160145 150 155 160

Ser Lys Asn Arg Val Pro Asp Ser Cys Cys Ile Asn Val Thr Val GlySer Lys Asn Arg Val Pro Asp Ser Cys Cys Ile Asn Val Thr Val Gly

165 170 175 165 170 175

Cys Gly Ile Asn Phe Asn Glu Lys Ala Ile His Lys Glu Gly Cys ValCys Gly Ile Asn Phe Asn Glu Lys Ala Ile His Lys Glu Gly Cys Val

180 185 190 180 185 190

Glu Lys Ile Gly Gly Trp Leu Arg Lys Asn Val Leu Val Val Ala AlaGlu Lys Ile Gly Gly Trp Leu Arg Lys Asn Val Leu Val Val Ala Ala

195 200 205 195 200 205

Ala Ala Leu Gly Ile Ala Phe Val Glu Val Leu Gly Ile Val Phe AlaAla Ala Leu Gly Ile Ala Phe Val Glu Val Leu Gly Ile Val Phe Ala

210 215 220 210 215 220

Cys Cys Leu Val Lys Ser Ile Arg Ser Gly Tyr Glu Val MetCys Cys Leu Val Lys Ser Ile Arg Ser Gly Tyr Glu Val Met

225 230 235225 230 235

<210> 12<210> 12

<211> 717<211> 717

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> CD63核苷酸序列<223> CD63 nucleotide sequence

<400> 12<400> 12

atggcggtgg aaggaggaat gaaatgtgtg aagttcttgc tctacgtcct cctgctggcc 60atggcggtgg aaggaggaat gaaatgtgtg aagttcttgc tctacgtcct cctgctggcc 60

ttttgcgcct gtgcagtggg actgattgcc gtgggtgtcg gggcacagct tgtcctgagt 120ttttgcgcct gtgcagtggg actgattgcc gtgggtgtcg gggcacagct tgtcctgagt 120

cagaccataa tccagggggc tacccctggc tctctgttgc cagtggtcat catcgcagtg 180cagaccataa tccagggggc tacccctggc tctctgttgc cagtggtcat catcgcagtg 180

ggtgtcttcc tcttcctggt ggcttttgtg ggctgctgcg gggcctgcaa ggagaactat 240ggtgtcttcc tcttcctggt ggcttttgtg ggctgctgcg gggcctgcaa ggagaactat 240

tgtcttatga tcacgtttgc catctttctg tctcttatca tgttggtgga ggtggccgca 300tgtcttatga tcacgtttgc catctttctg tctcttatca tgttggtgga ggtggccgca 300

gccattgctg gctatgtgtt tagagataag gtgatgtcag agtttaataa caacttccgg 360gccattgctg gctatgtgtt tagagataag gtgatgtcag agtttaataa caacttccgg 360

cagcagatgg agaattaccc gaaaaacaac cacactgctt cgatcctgga caggatgcag 420cagcagatgg agaattaccc gaaaaacaac cacactgctt cgatcctgga caggatgcag 420

gcagatttta agtgctgtgg ggctgctaac tacacagatt gggagaaaat cccttccatg 480gcagatttta agtgctgtgg ggctgctaac tacacagatt gggagaaaat cccttccatg 480

tcgaagaacc gagtccccga ctcctgctgc attaatgtta ctgtgggctg tgggattaat 540tcgaagaacc gagtccccga ctcctgctgc attaatgtta ctgtgggctg tgggattaat 540

ttcaacgaga aggcgatcca taaggagggc tgtgtggaga agattggggg ctggctgagg 600ttcaacgaga aggcgatcca taaggagggc tgtgtggaga agattggggg ctggctgagg 600

aaaaatgtgc tggtggtagc tgcagcagcc cttggaattg cttttgtcga ggttttggga 660aaaaatgtgc tggtggtagc tgcagcagcc cttggaattg cttttgtcga ggttttggga 660

attgtctttg cctgctgcct cgtgaagagt atcagaagtg gctacgaggt gatgtag 717attgtctttg cctgctgcct cgtgaagagt atcagaagtg gctacgaggt gatgtag 717

<210> 13<210> 13

<211> 236<211> 236

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> CD81氨基酸序列<223> CD81 amino acid sequence

<400> 13<400> 13

Met Gly Val Glu Gly Cys Thr Lys Cys Ile Lys Tyr Leu Leu Phe ValMet Gly Val Glu Gly Cys Thr Lys Cys Ile Lys Tyr Leu Leu Phe Val

1 5 10 151 5 10 15

Phe Asn Phe Val Phe Trp Leu Ala Gly Gly Val Ile Leu Gly Val AlaPhe Asn Phe Val Phe Trp Leu Ala Gly Gly Val Ile Leu Gly Val Ala

20 25 30 20 25 30

Leu Trp Leu Arg His Asp Pro Gln Thr Thr Asn Leu Leu Tyr Leu GluLeu Trp Leu Arg His Asp Pro Gln Thr Thr Asn Leu Leu Tyr Leu Glu

35 40 45 35 40 45

Leu Gly Asp Lys Pro Ala Pro Asn Thr Phe Tyr Val Gly Ile Tyr IleLeu Gly Asp Lys Pro Ala Pro Asn Thr Phe Tyr Val Gly Ile Tyr Ile

50 55 60 50 55 60

Leu Ile Ala Val Gly Ala Val Met Met Phe Val Gly Phe Leu Gly CysLeu Ile Ala Val Gly Ala Val Met Met Met Phe Val Gly Phe Leu Gly Cys

65 70 75 8065 70 75 80

Tyr Gly Ala Ile Gln Glu Ser Gln Cys Leu Leu Gly Thr Phe Phe ThrTyr Gly Ala Ile Gln Glu Ser Gln Cys Leu Leu Gly Thr Phe Phe Thr

85 90 95 85 90 95

Cys Leu Val Ile Leu Phe Ala Cys Glu Val Ala Ala Gly Ile Trp GlyCys Leu Val Ile Leu Phe Ala Cys Glu Val Ala Ala Gly Ile Trp Gly

100 105 110 100 105 110

Phe Val Asn Lys Asp Gln Ile Ala Lys Asp Val Lys Gln Phe Tyr AspPhe Val Asn Lys Asp Gln Ile Ala Lys Asp Val Lys Gln Phe Tyr Asp

115 120 125 115 120 125

Gln Ala Leu Gln Gln Ala Val Val Asp Asp Asp Ala Asn Asn Ala LysGln Ala Leu Gln Gln Ala Val Val Asp Asp Asp Ala Asn Asn Ala Lys

130 135 140 130 135 140

Ala Val Val Lys Thr Phe His Glu Thr Leu Asp Cys Cys Gly Ser SerAla Val Val Lys Thr Phe His Glu Thr Leu Asp Cys Cys Gly Ser Ser

145 150 155 160145 150 155 160

Thr Leu Thr Ala Leu Thr Thr Ser Val Leu Lys Asn Asn Leu Cys ProThr Leu Thr Ala Leu Thr Thr Ser Val Leu Lys Asn Asn Leu Cys Pro

165 170 175 165 170 175

Ser Gly Ser Asn Ile Ile Ser Asn Leu Phe Lys Glu Asp Cys His GlnSer Gly Ser Asn Ile Ile Ser Asn Leu Phe Lys Glu Asp Cys His Gln

180 185 190 180 185 190

Lys Ile Asp Asp Leu Phe Ser Gly Lys Leu Tyr Leu Ile Gly Ile AlaLys Ile Asp Asp Leu Phe Ser Gly Lys Leu Tyr Leu Ile Gly Ile Ala

195 200 205 195 200 205

Ala Ile Val Val Ala Val Ile Met Ile Phe Glu Met Ile Leu Ser MetAla Ile Val Val Ala Val Ile Met Ile Phe Glu Met Ile Leu Ser Met

210 215 220 210 215 220

Val Leu Cys Cys Gly Ile Arg Asn Ser Ser Val TyrVal Leu Cys Cys Gly Ile Arg Asn Ser Ser Val Tyr

225 230 235225 230 235

<210> 14<210> 14

<211> 711<211> 711

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> CD81核苷酸序列<223> CD81 nucleotide sequence

<400> 14<400> 14

atgggagtgg agggctgcac caagtgcatc aagtacctgc tcttcgtctt caatttcgtc 60atgggagtgg agggctgcac caagtgcatc aagtacctgc tcttcgtctt caatttcgtc 60

ttctggctgg ctggaggcgt gatcctgggt gtggccctgt ggctccgcca tgacccgcag 120ttctggctgg ctggaggcgt gatcctgggt gtggccctgt ggctccgcca tgacccgcag 120

accaccaacc tcctgtatct ggagctggga gacaagcccg cgcccaacac cttctatgta 180accaccaacc tcctgtatct ggagctggga gacaagcccg cgcccaacac cttctatgta 180

ggcatctaca tcctcatcgc tgtgggcgct gtcatgatgt tcgttggctt cctgggctgc 240ggcatctaca tcctcatcgc tgtgggcgct gtcatgatgt tcgttggctt cctgggctgc 240

tacggggcca tccaggaatc ccagtgcctg ctggggacgt tcttcacctg cctggtcatc 300tacggggcca tccaggaatc ccagtgcctg ctggggacgt tcttcacctg cctggtcatc 300

ctgtttgcct gtgaggtggc cgccggcatc tggggctttg tcaacaagga ccagatcgcc 360ctgtttgcct gtgaggtggc cgccggcatc tggggctttg tcaacaagga ccagatcgcc 360

aaggatgtga agcagttcta tgaccaggcc ctacagcagg ccgtggtgga tgatgacgcc 420aaggatgtga agcagttcta tgaccaggcc ctacagcagg ccgtggtgga tgatgacgcc 420

aacaacgcca aggctgtggt gaagaccttc cacgagacgc ttgactgctg tggctccagc 480aacaacgcca aggctgtggt gaagaccttc cacgagacgc ttgactgctg tggctccagc 480

acactgactg ctttgaccac ctcagtgctc aagaacaatt tgtgtccctc gggcagcaac 540acactgactg ctttgaccac ctcagtgctc aagaacaatt tgtgtccctc gggcagcaac 540

atcatcagca acctcttcaa ggaggactgc caccagaaga tcgatgacct cttctccggg 600atcatcagca acctcttcaa ggaggactgc caccagaaga tcgatgacct cttctccggg 600

aagctgtacc tcatcggcat tgctgccatc gtggtcgctg tgatcatgat cttcgagatg 660aagctgtacc tcatcggcat tgctgccatc gtggtcgctg tgatcatgat cttcgagatg 660

atcctgagca tggtgctgtg ctgtggcatc cggaacagct ccgtgtactg a 711atcctgagca tggtgctgtg ctgtggcatc cggaacagct ccgtgtactg a 711

<210> 15<210> 15

<211> 227<211> 227

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> CD9氨基酸序列<223> CD9 amino acid sequence

<400> 15<400> 15

Met Pro Val Lys Gly Gly Thr Lys Cys Ile Lys Tyr Leu Leu Phe GlyMet Pro Val Lys Gly Gly Thr Lys Cys Ile Lys Tyr Leu Leu Phe Gly

1 5 10 151 5 10 15

Phe Asn Phe Ile Phe Trp Leu Ala Gly Ile Ala Val Leu Ala Ile GlyPhe Asn Phe Ile Phe Trp Leu Ala Gly Ile Ala Val Leu Ala Ile Gly

20 25 30 20 25 30

Leu Trp Leu Arg Phe Asp Ser Gln Thr Lys Ser Ile Phe Glu Gln GluLeu Trp Leu Arg Phe Asp Ser Gln Thr Lys Ser Ile Phe Glu Gln Glu

35 40 45 35 40 45

Thr Asn Asn Asn Asn Ser Ser Phe Tyr Thr Gly Val Tyr Ile Leu IleThr Asn Asn Asn Asn Ser Ser Phe Tyr Thr Gly Val Tyr Ile Leu Ile

50 55 60 50 55 60

Gly Ala Gly Ala Leu Met Met Leu Val Gly Phe Leu Gly Cys Cys GlyGly Ala Gly Ala Leu Met Met Leu Val Gly Phe Leu Gly Cys Cys Gly

65 70 75 8065 70 75 80

Ala Val Gln Glu Ser Gln Cys Met Leu Gly Leu Phe Phe Gly Phe LeuAla Val Gln Glu Ser Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu

85 90 95 85 90 95

Leu Val Ile Phe Ala Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr SerLeu Val Ile Phe Ala Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr Ser

100 105 110 100 105 110

His Lys Asp Glu Val Ile Lys Glu Val Gln Glu Phe Tyr Lys Asp ThrHis Lys Asp Glu Val Ile Lys Glu Val Gln Glu Phe Tyr Lys Asp Thr

115 120 125 115 120 125

Tyr Asn Lys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu LysTyr Asn Lys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys

130 135 140 130 135 140

Ala Ile His Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val GluAla Ile His Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val Glu

145 150 155 160145 150 155 160

Gln Phe Ile Ser Asp Ile Cys Pro Lys Lys Asp Val Leu Glu Thr PheGln Phe Ile Ser Asp Ile Cys Pro Lys Lys Asp Val Leu Glu Thr Phe

165 170 175 165 170 175

Thr Val Lys Ser Cys Pro Asp Ala Ile Lys Glu Val Phe Asp Asn LysThr Val Lys Ser Cys Pro Asp Ala Ile Lys Glu Val Phe Asp Asn Lys

180 185 190 180 185 190

Phe His Ile Ile Gly Ala Val Gly Ile Gly Ile Ala Val Val Met IlePhe His Ile Ile Gly Ala Val Gly Ile Gly Ile Ala Val Val Met Ile

195 200 205 195 200 205

Phe Gly Met Ile Phe Ser Met Ile Leu Cys Cys Ala Ile Arg Arg AsnPhe Gly Met Ile Phe Ser Met Ile Leu Cys Cys Ala Ile Arg Arg Asn

210 215 220 210 215 220

Arg Glu MetArg Glu Met

225225

<210> 16<210> 16

<211> 687<211> 687

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> CD9核苷酸序列<223> CD9 nucleotide sequence

<400> 16<400> 16

atgccggtca aaggaggcac caagtgcatc aaatacctgc tgttcggatt taacttcatc 60atgccggtca aaggaggcac caagtgcatc aaatacctgc tgttcggatt taacttcatc 60

ttctggcttg ccgggattgc tgtccttgcc attggactat ggctccgatt cgactctcag 120ttctggcttg ccgggattgc tgtccttgcc attggactat ggctccgatt cgactctcag 120

accaagagca tcttcgagca agaaactaat aataataatt ccagcttcta cacaggagtc 180accaagagca tcttcgagca agaaactaat aataataatt ccagcttcta cacaggagtc 180

tatattctga tcggagccgg cgccctcatg atgctggtgg gcttcctggg ctgctgcggg 240tatattctga tcggagccgg cgccctcatg atgctggtgg gcttcctggg ctgctgcggg 240

gctgtgcagg agtcccagtg catgctggga ctgttcttcg gcttcctctt ggtgatattc 300gctgtgcagg agtcccagtg catgctggga ctgttcttcg gcttcctctt ggtgatattc 300

gccattgaaa tagctgcggc catctgggga tattcccaca aggatgaggt gattaaggaa 360gccattgaaa tagctgcggc catctgggga tattcccaca aggatgaggt gattaaggaa 360

gtccaggagt tttacaagga cacctacaac aagctgaaaa ccaaggatga gccccagcgg 420gtccaggagt tttacaagga cacctacaac aagctgaaaa ccaaggatga gccccagcgg 420

gaaacgctga aagccatcca ctatgcgttg aactgctgtg gtttggctgg gggcgtggaa 480gaaacgctga aagccatcca ctatgcgttg aactgctgtg gtttggctgg gggcgtggaa 480

cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac cgtgaagtcc 540cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac cgtgaagtcc 540

tgtcctgatg ccatcaaaga ggtcttcgac aataaattcc acatcatcgg cgcagtgggc 600tgtcctgatg ccatcaaaga ggtcttcgac aataaattcc acatcatcgg cgcagtgggc 600

atcggcattg ccgtggtcat gatatttggc atgatcttca gtatgatctt gtgctgtgct 660atcggcattg ccgtggtcat gatatttggc atgatcttca gtatgatctt gtgctgtgct 660

atccgcagga accgcgagat ggtctag 687atccgcagga accgcgagat ggtctag 687

<210> 17<210> 17

<211> 243<211> 243

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220> <220>

<223> TP01氨基酸序列<223> TP01 amino acid sequence

<400> 17<400> 17

Met Pro Ala Pro Ala Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu CysMet Pro Ala Pro Ala Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys

1 5 10 151 5 10 15

Ala Pro Ser Leu Leu Val Ala Leu Ala Ile Cys Ser Leu Ala Pro CysAla Pro Ser Leu Leu Val Ala Leu Ala Ile Cys Ser Leu Ala Pro Cys

20 25 30 20 25 30

His Ala Gly Gly Leu Cys Gly Gly Ile Ser Gly Gly Val Ala Gly AlaHis Ala Gly Gly Leu Cys Gly Gly Ile Ser Gly Gly Val Ala Gly Ala

35 40 45 35 40 45

Val Pro Pro Ser Thr Thr Cys Thr Cys Leu Leu Gly Thr Ala Gly AlaVal Pro Pro Ser Thr Thr Cys Thr Cys Leu Leu Gly Thr Ala Gly Ala

50 55 60 50 55 60

His Cys Gly Thr Leu Cys Val Gly Pro Leu Gly Leu Gly Ala Gly AlaHis Cys Gly Thr Leu Cys Val Gly Pro Leu Gly Leu Gly Ala Gly Ala

65 70 75 8065 70 75 80

Ile Ala Ala Ser Gly Ile Ala Ala Ser Ser Val Ala Val Thr Pro LeuIle Ala Ala Ser Gly Ile Ala Ala Ser Ser Val Ala Val Thr Pro Leu

85 90 95 85 90 95

Gly Leu Gly His Thr Val Pro Gly Leu Ala Ala Leu Ala Ala Ala GlyGly Leu Gly His Thr Val Pro Gly Leu Ala Ala Leu Ala Ala Ala Gly

100 105 110 100 105 110

Met Val Ala Ala Thr Thr Pro Ser Ser Ala Ala Ala Ala Pro Thr IleMet Val Ala Ala Thr Thr Pro Ser Ser Ala Ala Ala Ala Pro Thr Ile

115 120 125 115 120 125

Gly Val Ala Leu Leu Ala Ala Met Thr Val Thr Gly Val Val Thr GlyGly Val Ala Leu Leu Ala Ala Met Thr Val Thr Gly Val Val Thr Gly

130 135 140 130 135 140

Gly Ala Ser Ala Leu Ala Ser His Gly Thr Leu Leu Ala Pro Leu ValGly Ala Ser Ala Leu Ala Ser His Gly Thr Leu Leu Ala Pro Leu Val

145 150 155 160145 150 155 160

Ala Thr Ser Leu Ala Gly His Gly Pro Ala Pro Ile His Ala Val AlaAla Thr Ser Leu Ala Gly His Gly Pro Ala Pro Ile His Ala Val Ala

165 170 175 165 170 175

Leu Leu His Leu Gly Pro Val Gly Ala Thr Ala Leu Ala Ala Val HisLeu Leu His Leu Gly Pro Val Gly Ala Thr Ala Leu Ala Ala Val His

180 185 190 180 185 190

Val Ala Leu Pro Gly Thr Pro Val Gly Ala Gly Thr Val Ala Leu ThrVal Ala Leu Pro Gly Thr Pro Val Gly Ala Gly Thr Val Ala Leu Thr

195 200 205 195 200 205

Pro Thr Ser Cys His Thr Ala Cys Thr Leu Ala Pro Gly Leu Leu GlyPro Thr Ser Cys His Thr Ala Cys Thr Leu Ala Pro Gly Leu Leu Gly

210 215 220 210 215 220

Cys Gly Leu Ala Ala Ala Leu Ala Ala Leu Ala Ala Gly Ala Ala AlaCys Gly Leu Ala Ala Ala Leu Ala Ala Leu Ala Ala Gly Ala Ala Ala

225 230 235 240225 230 235 240

Ala Gly GlyAla Gly Gly

<210> 18<210> 18

<211> 732<211> 732

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220> <220>

<223> TP01核苷酸序列<223> TP01 Nucleotide Sequence

<400> 18<400> 18

atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 60atgccgcgcc cccgcctgct ggccgcgctg tgcggcgcgc tgctctgcgc ccccagcctc 60

ctcgtcgccc tggatatctg ttccaaaaac ccctgccaca acggtggttt atgcgaggag 120ctcgtcgccc tggatatctg ttccaaaaac ccctgccaca acggtggttt atgcgaggag 120

atttcccaag aagtgcgagg agatgtcttc ccctcgtaca cctgcacgtg ccttaagggc 180atttcccaag aagtgcgagg agatgtcttc ccctcgtaca cctgcacgtg ccttaagggc 180

tacgcgggca accactgtga gacgaaatgt gtcgagccac tgggcctgga gaatgggaac 240tacgcgggca accactgtga gacgaaatgt gtcgagccac tgggcctgga gaatgggaac 240

attgccaact cacagatcgc cgcctcgtct gtgcgtgtga ccttcttggg tttgcagcat 300attgccaact cacagatcgc cgcctcgtct gtgcgtgtga ccttcttggg tttgcagcat 300

tgggtcccgg agctggcccg cctgaaccgc gcaggcatgg tcaatgcctg gacacccagc 360tgggtcccgg agctggcccg cctgaaccgc gcaggcatgg tcaatgcctg gacacccagc 360

agcaatgacg ataacccctg gatccaggtg aacctgctgc ggaggatgtg ggtaacaggt 420agcaatgacg ataacccctg gatccaggtg aacctgctgc ggaggatgtg ggtaacaggt 420

gtggtgacgc agggtgccag ccgcttggcc agtcatgagt acctgaaggc cttcaaggtg 480gtggtgacgc agggtgccag ccgcttggcc agtcatgagt acctgaaggc cttcaaggtg 480

gcctacagcc ttaatggaca cgaattcgat ttcatccatg atgttaataa aaaacacaag 540gcctacagcc ttaatggaca cgaattcgat ttcatccatg atgttaataa aaaacacaag 540

gagtttgtgg gtaactggaa caaaaacgcg gtgcatgtca acctgtttga gacccctgtg 600gagtttgtgg gtaactggaa caaaaacgcg gtgcatgtca acctgtttga gacccctgtg 600

gaggctcagt acgtgagatt gtaccccacg agctgccaca cggcctgcac tctgcgcttt 660gaggctcagt acgtgagatt gtaccccacg agctgccaca cggcctgcac tctgcgcttt 660

gagctactgg gctgtgagct gaacgcaagg aaggcagact tgaggcgagg agcagatgac 720gagctactgg gctgtgagct gaacgcaagg aaggcagact tgaggcgagg agcagatgac 720

agagagcagt aa 732agagagcagt aa 732

<210> 19<210> 19

<211> 25<211> 25

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> EGFP-F<223> EGFP-F

<400> 19<400> 19

ataaaaggta ccatggtgag caagg 25ataaaaggta ccatggtgag caagg 25

<210> 20<210> 20

<211> 28<211> 28

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<223> EGFP-R<223> EGFP-R

<400> 20<400> 20

ataaaaggat ccttacttgt acagctcg 28ataaaaggat ccttacttgt acagctcg 28

<210> 21<210> 21

<211> 42<211> 42

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220> <220>

<223> F-CD63-L7Ae<223> F-CD63-L7Ae

<400> 21<400> 21

ataaaagcta gcatggcggt ggaaggagga atgaaatgtg tg 42ataaaagcta gcatggcggt ggaaggagga atgaaatgtg tg 42

<210> 22 <210> 22

<211> 43<211> 43

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220> <220>

<223> R-CD63-L7Ae<223> R-CD63-L7Ae

<400> 22<400> 22

ataaaaggat ccttagtggt ggtggtggtg gtgcttctga agg 43ataaaaggat ccttagtggt ggtggtggtg gtgcttctga agg 43

<210> 23 <210> 23

<211> 45<211> 45

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220> <220>

<223> Linker<223> Linker

<400> 23 <400> 23

ggtggaggtg gcagcggagg aggtgggtcc ggcggtggag gaagc 45ggtggaggtg gcagcggagg aggtgggtcc ggcggtggag gaagc 45

Claims (7)

Translated fromChinese

1.一种装载shRNA的外泌体的构建方法，其特征在于：包括如下步骤：1. a construction method of the exosome of loading shRNA, is characterized in that: comprise the steps:(1)设计、合成可以有效沉默靶基因的shRNA序列；将所述的shRNA序列克隆入表达载体，得到重组表达载体1；所述的shRNA序列包括依次连接的靶序列、茎环结构序列和靶序列的互补序列；(1) Design and synthesize an shRNA sequence that can effectively silence the target gene; clone the shRNA sequence into an expression vector to obtain a recombinant expression vector 1; the shRNA sequence includes a target sequence, a stem-loop structure sequence and a target sequence connected in sequence the complement of the sequence;(2)将能与所述的茎环结构特异性结合的蛋白的基因与外泌体膜蛋白的基因连接，得到融合基因序列，再将所得的融合基因序列克隆入表达载体，得到重组表达载体2；(2) connecting the gene of the protein that can specifically bind to the stem-loop structure with the gene of the exosome membrane protein to obtain a fusion gene sequence, and then clone the obtained fusion gene sequence into an expression vector to obtain a recombinant expression vector 2;(3)将步骤(1)得到的重组表达载体1和步骤(2)得到的重组表达载体2同时转染细胞，培养转染后细胞，收集外泌体，即获得所述的装载shRNA的外泌体；(3) Simultaneously transfect the cells with the recombinant expression vector 1 obtained in step (1) and the recombinant expression vector 2 obtained in step (2), culture the transfected cells, and collect exosomes to obtain the exosomes loaded with shRNA. exosomes;步骤(1)中所述的茎环结构序列为GCTGACCCGAAAGGGCGTGATGC；The stem-loop structure sequence described in step (1) is GCTGACCCGAAAGGGCGTGATGC;步骤(2)中所述的能与所述的茎环结构特异性结合的蛋白选自RNA结合蛋白L7Ae、NHP2核糖核蛋白样蛋白1、人核蛋白P56和人核蛋白P58中的至少一种；The protein that can specifically bind to the stem-loop structure described in step (2) is selected from at least one of RNA-binding protein L7Ae, NHP2 ribonucleoprotein-like protein 1, human nuclear protein P56 and human nuclear protein P58 ;步骤(2)中所述的外泌体膜蛋白为CD63、CD81、CD9和TP01中的至少一种。The exosomal membrane protein described in step (2) is at least one of CD63, CD81, CD9 and TP01.2.根据权利要求1所述的装载shRNA的外泌体的构建方法，其特征在于：2. the construction method of the exosome of loading shRNA according to claim 1, is characterized in that:所述的RNA结合蛋白L7Ae的氨基酸序列如SEQ ID NO.3所示；The amino acid sequence of the RNA-binding protein L7Ae is shown in SEQ ID NO.3;所述的NHP2核糖核蛋白样蛋白1的氨基酸序列如SEQ ID NO.5所示；The amino acid sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO.5;所述的人核蛋白P56的氨基酸序列如SEQ ID NO.7所示；The amino acid sequence of described human nucleoprotein P56 is shown in SEQ ID NO.7;所述的人核蛋白P58的氨基酸序列如SEQ ID NO.9所示；The amino acid sequence of the human nucleoprotein P58 is shown in SEQ ID NO.9;所述的CD63的氨基酸序列如SEQ ID NO.11所示；The amino acid sequence of the CD63 is shown in SEQ ID NO.11;所述的CD81的氨基酸序列如SEQ ID NO.13所示；The amino acid sequence of the CD81 is shown in SEQ ID NO.13;所述的CD9的氨基酸序列如SEQ ID NO.15所示；The amino acid sequence of the CD9 is shown in SEQ ID NO.15;所述的TP01的氨基酸序列如SEQ ID NO.17所示。The amino acid sequence of TP01 is shown in SEQ ID NO.17.3.根据权利要求1所述的装载shRNA的外泌体的构建方法，其特征在于：3. the construction method of the exosome of loading shRNA according to claim 1, is characterized in that:所述的RNA结合蛋白L7Ae的核苷酸序列如SEQ ID NO.4所示；The nucleotide sequence of the RNA binding protein L7Ae is shown in SEQ ID NO.4;所述的NHP2核糖核蛋白样蛋白1的核苷酸序列如SEQ ID NO.6所示；The nucleotide sequence of the NHP2 ribonucleoprotein-like protein 1 is shown in SEQ ID NO.6;所述的人核蛋白P56的核苷酸序列如SEQ ID NO.8所示；The nucleotide sequence of the human nucleoprotein P56 is shown in SEQ ID NO.8;所述的人核蛋白P58的核苷酸序列如SEQ ID NO.10所示；The nucleotide sequence of the human nucleoprotein P58 is shown in SEQ ID NO.10;所述的CD63的核苷酸序列如SEQ ID NO.12所示；The nucleotide sequence of the CD63 is shown in SEQ ID NO.12;所述的CD81的核苷酸序列如SEQ ID NO.14所示；The nucleotide sequence of the CD81 is shown in SEQ ID NO.14;所述的CD9的核苷酸序列如SEQ ID NO 16所示；The nucleotide sequence of the CD9 is shown in SEQ ID NO 16;所述的TP01的核苷酸序列如SEQ ID NO.18所示。The nucleotide sequence of TP01 is shown in SEQ ID NO.18.4.根据权利要求1所述的装载shRNA的外泌体的构建方法，其特征在于：4. the construction method of the exosome of loading shRNA according to claim 1, is characterized in that:步骤(2)中所述的连接为通过接头连接，连接的顺序为蛋白A的基因片段-接头-外泌体膜蛋白基因片段；The connection described in the step (2) is to be connected by a joint, and the order of the connection is the gene fragment of protein A-joiner-exosome membrane protein gene fragment;所述的接头的核苷酸序列为：The nucleotide sequence of the linker is:GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC。GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGC.5.根据权利要求1所述的装载shRNA的外泌体的构建方法，其特征在于：5. the construction method of the exosome of loading shRNA according to claim 1, is characterized in that:步骤(1)中所述的表达载体为pRNAT-U6.1/Neo；The expression vector described in step (1) is pRNAT-U6.1/Neo;步骤(2)中所述的表达载体为pCDNA3.4。The expression vector described in step (2) is pCDNA3.4.6.根据权利要求1所述的装载shRNA的外泌体的构建方法，其特征在于：6. the construction method of the exosome of loading shRNA according to claim 1 is characterized in that:步骤(3)中，在转染细胞时，重组表达载体1和重组表达载体2的数量比为1～2:1～2。In step (3), when transfecting cells, the quantity ratio of recombinant expression vector 1 and recombinant expression vector 2 is 1-2:1-2.7.一种装载shRNA的外泌体，其特征在于：根据权利要求1～6任一项所述的构建方法得到。7. An exosome loaded with shRNA, characterized in that: obtained according to the construction method described in any one of claims 1 to 6.

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