技术领域technical field
本发明属生物制药技术领域，涉及人粒细胞集落刺激因子的重组融合蛋白，具体涉及一种长效的重组人粒细胞集落刺激因子及其制备方法和用途。本发明在保持人粒细胞集落刺激因子的原有活性的前提下，延长该蛋白质在体内的半衰期，进一步用于制备抗放化疗或其他原因造成的中性粒细胞减少症以及骨髓抑制的药物。The invention belongs to the technical field of biopharmaceuticals, and relates to a recombinant fusion protein of human granulocyte colony-stimulating factor, in particular to a long-acting recombinant human granulocyte colony-stimulating factor and its preparation method and application. On the premise of maintaining the original activity of human granulocyte colony-stimulating factor, the present invention prolongs the half-life of the protein in vivo, and is further used to prepare drugs for resisting neutropenia and bone marrow suppression caused by radiotherapy, chemotherapy or other reasons.
背景技术Background technique
据报道，恶性肿瘤是当前严重影响人类健康、威胁人类生命的主要疾病之一。据统计，2007年全球有760万人死于癌症，其中有大约24％发生在中国，并呈现出逐步年轻化的趋势。目前癌症的治疗仍以手术、放疗和化疗为主要治疗手段，其中，放疗和化疗会大量杀伤人体正常的白细胞，造成病人骨髓抑制、免疫力低下，容易引发各种感染等并发症。According to reports, malignant tumors are currently one of the major diseases that seriously affect human health and threaten human life. According to statistics, 7.6 million people died of cancer in the world in 2007, about 24% of which occurred in China, showing a gradual trend of younger people. At present, cancer treatment still relies on surgery, radiotherapy and chemotherapy as the main treatment methods. Among them, radiotherapy and chemotherapy will kill a large number of normal white blood cells in the human body, causing bone marrow suppression and low immunity in patients, and easily causing various infections and other complications.
研究显示，重组人粒细胞集落刺激因子(Recombinanthumangranulocytecolonystimulatingfactor,rhG-CSF)与人体自身分泌的粒细胞集落刺激因子相似或相同，主要作用于中性粒细胞系造血细胞的增殖、分化和活化；能刺激骨髓造血祖细胞中中性粒细胞集落的形成，延长成熟中性粒细胞的存活时间(RobertsAW.G-CSF.Akeyregulatorofneutrophilproduction,butthat”snotall！GrowthFactors.2005；23(1):33–41；DemetriGD,GriffinJD.Granulocytecolonystimulatingfactoranditsreceptor.Blood.1991；78(11):2791-2808；DeJesusCE,EgenJ,MetzgerM,etal.Transientneutropeniaaftergranulocyte-colonystimulatingfactoradministrationisassociatedwithneutrophilaccumulationinpulmonaryvasculature.ExperimentalHematology.2011；39(2):142-150.)，临床上主要用于治疗肿瘤化疗后的中性粒细胞减少症等疾病，其应用量巨大。目前国内主要销售的该类药物有：瑞白，惠尔血等，至2010年，该类药物的销售额已经达到近10亿元。但临床实践显示，该类药物在体内半衰期较短，通常只有4-6小时(YoshidaT1,NakamuraS,OhtakeS,OkafujiK,KobayashiK,KondoK,KannoM,MatanoS,MatsudaT,KanaiM,etal.Effectofgranulocytecolony-stimulatingfactoronneutropeniaduetochemotherapyfornon-Hodgkin'slymphoma.Cancer.1990；66(9):1904-9.)，因此，患者需要每天注射以维持药效，病人耐受较差。Studies have shown that recombinant human granulocyte colony stimulating factor (Recombinanthumangranulocyte colony stimulating factor, rhG-CSF) is similar or identical to the granulocyte colony stimulating factor secreted by the human body, mainly acts on the proliferation, differentiation and activation of neutrophil hematopoietic cells; can stimulate Formation of neutrophil colonies in bone marrow hematopoietic progenitors prolongs survival of mature neutrophils (RobertsAW. GriffinJD.Granulocytecolonystimulatingfactoranditsreceptor.Blood.1991；78(11):2791-2808；DeJesusCE,EgenJ,MetzgerM,etal.Transientneutropeniaaftergranulocyte-colonystimulatingfactoradministrationisassociatedwithneutrophilaccumulationinpulmonaryvasculature.ExperimentalHematology.2011；39(2):142-150.)，临床上主要用于治疗Neutropenia and other diseases after tumor chemotherapy are widely used. At present, the main domestic sales of such drugs are: Ruibai, Huierxue, etc., and by 2010, the sales of such drugs have reached nearly 10 However, clinical practice shows that the half-life of such drugs in the body is short, usually only 4-6 hours (YoshidaT1, NakamuraS, OhtakeS, OkafujiK, KobayashiK, KondoK, KannoM, MatanoS, MatsudaT, KanaiM, et al. Effect of granulocyte colony-stimulating factor on neutropenia due to chemotherapy for non- Hodgkin'slymphoma.Cancer.1990; 66(9):1904-9.), therefore, patients need daily injections to maintain drug efficacy, and the patient's tolerance is poor.
有研究公开了对重组人粒细胞集落刺激因子的长效化改造中，制备了聚乙二醇修饰的重组人粒细胞集落刺激因子，其中，长效重组人粒细胞集落刺激因子产品Neulasta(pegfilgrastim)是PEG化的G-CSF,于2002年被美国FDA批准上市；所述Neulasta在体内半衰期和药效方面显示了其较同类产品有较明显的提升，患者只需在每次化疗前注射一次即可在化疗中保持原本的粒细胞水平，极大地减少了患者的痛苦；目前，该药物价格昂贵，每支售价5000美元，国内尚无该进口药，在研的G-CSF-Fc等融合蛋白类药物尚处于临床或临床前阶段。Some studies have disclosed that in the long-acting transformation of recombinant human granulocyte colony-stimulating factor, polyethylene glycol-modified recombinant human granulocyte-colony-stimulating factor was prepared, wherein the long-acting recombinant human granulocyte-colony-stimulating factor product Neulasta (pegfilgrastim ) is PEGylated G-CSF, which was approved for marketing by the US FDA in 2002; the Neulasta in vivo half-life and drug efficacy have shown a significant improvement compared with similar products, and patients only need to inject once before each chemotherapy It can maintain the original level of granulocytes during chemotherapy, which greatly reduces the pain of patients; at present, the drug is expensive, with a price of 5,000 US dollars per tube, and there is no such imported drug in China. G-CSF-Fc, etc. Fusion protein drugs are still in the clinical or preclinical stage.
CTP(Carboxyl-TerminalPeptideofHumanChorionicGonadotropinβ-Subunit)是由人绒毛膜促性腺激素(hCG)β亚基端118位到145位的氨基酸所组成的短肽。研究发现，hCG的体内半衰期相比其他生长激素类的要长，而HCG的β亚基C端的CTP在其中起了相当重要的作用(BoulouxPM,HandelsmanDJ,JockenhvelF,etal.FirsthumanexposuretoFSH-CTPinhypogonadotrophichypogonadalmales.Humanreproduction.2001；16(8):1592-1597；DuijkersIJ,KlippingC,BoerrigterPJ,etal.Singledosepharmacokineticsandeffectsonfolliculargrowthandserumhormonesofalong-actingrecombinantFSHpreparation(FSH-CTP)inhealthypituitary-suppressedfemales.Humanreproduction.2002；17(8):1987-1993；MatzukMM,HsuehAJ,LapoltP,etal.Thebiologicalroleofthecarboxyl-terminalextensionofhumanchorionicgonadotropin[corrected]beta-subunit.Endocrinology.1990；126(1):376-383.)，表明CTP融合蛋白可能比野生型蛋白质具有更长的半衰期。还有研究对CTP-FSH(卵泡刺激素)CTP-TSHβ(促甲状腺激素)研究发现(FaresFA,SuganumaN,NishimoriK,etal.Designofalong-actingfollitropinagonistbyfusingtheCterminalsequenceofthechorionicgonadotropinβ-subunittothefollitropinβ-subunit.ProcNatlAcadSciUSA1992；89(10):4304–4308；LaPoltPS,NishimoriK,FaresFA,PerlasE,BoimeI,HsuehAJW.Enhancedstimulationoffolliclematurationandovulatorypotentialbylongactingfollicle-stimulatinghormoneagonistswithextendedcarboxyl-terminalpeptides.Endocrinology.1992；131(6):2514–2520)，CTP-hCGα(FuruhashiM,ShikoneT,FaresFA,SugaharaT,HsuehAJ,BoimeI.Fusingthecarboxy-terminalpeptideofthechorionicgonadotropin(CG)β-subunittothecommonβ-subunit:retentionofOlinkedglycosylationandenhancedinvivobioactivityofchimerichumanCG.MolEndocrinol.1995；9(1):54–63)，(JoshiL,MurataY,WondisfordFE,SzkudlinskiMW,DesaiR,WeintraubBD.Recombinantthyrotropincontainingaβ-subunitchimerawiththehumanchorionicgonadotropin-βcarboxyterminusisbiologicallyactive,withaprolongedplasmahalf-life:roleofcarbohydrateinbioactivityandmetabolicclearance.Endocrinology.1995；136(9):3839–3848)，重组CTP融合蛋白的生物活性及其它性质和野生型蛋白质相比几乎没有差异，但是半衰期则相对要长得多；证明了重组CTP融合蛋白不但具有良好的生物学活性，而且还具有较长的体内半衰期。CTP (Carboxyl-Terminal Peptide of Human Chorionic Gonadotropin β-Subunit) is a short peptide composed of amino acids from position 118 to position 145 of the β subunit of human chorionic gonadotropin (hCG). Studies have found that the in vivo half-life of hCG is longer than that of other growth hormones, and the CTP at the C-terminal of the β subunit of HCG plays a very important role in it (BoulouxPM, HandelsmanDJ, JockenhvelF, et al.FirsthumanexposuretoFSH-CTPinhypogonadotrophichypogonadalmales.Humanreproduction.2001 ；16(8):1592-1597；DuijkersIJ,KlippingC,BoerrigterPJ,etal.Singledosepharmacokineticsandeffectsonfolliculargrowthandserumhormonesofalong-actingrecombinantFSHpreparation(FSH-CTP)inhealthypituitary-suppressedfemales.Humanreproduction.2002；17(8):1987-1993；MatzukMM,HsuehAJ,LapoltP,etal . The biological role of the carboxyl-terminal extension of human chorionic gonadotropin [corrected] beta-subunit. Endocrinology. 1990; 126 (1): 376-383.), indicating that the CTP fusion protein may have a longer half-life than the wild-type protein. There are also studies on CTP-FSH (follicle stimulating hormone) and CTP-TSHβ (thyroid stimulating hormone) (FaresFA, SuganumaN, NishimoriK, et al. Design of along-acting follitropinagonist by fusing the Cterminal sequence of the chorionic gonadotropinβ-subunit to the follitropinβ-subunit. ProcNatlAcadSciUSA1992; 408-4); 408 (1 LaPoltPS,NishimoriK,FaresFA,PerlasE,BoimeI,HsuehAJW.Enhancedstimulationoffolliclematurationandovulatorypotentialbylongactingfollicle-stimulatinghormoneagonistswithextendedcarboxyl-terminalpeptides.Endocrinology.1992；131(6):2514–2520)，CTP-hCGα(FuruhashiM,ShikoneT,FaresFA,SugaharaT,HsuehAJ,BoimeI.Fusingthecarboxy- terminalpeptideofthechorionicgonadotropin(CG)β-subunittothecommonβ-subunit:retentionofOlinkedglycosylationandenhancedinvivobioactivityofchimerichumanCG.MolEndocrinol.1995；9(1):54–63)，(JoshiL,MurataY,WondisfordFE,SzkudlinskiMW,DesaiR,WeintraubBD.Recombinantthyrotropincontainingaβ-subunitchimerawiththehumanchorionicgonadotropin-βcarboxyterminusisbiologicallyactive,withaprolongedplasmahalf-life:roleofcarbohydrateinbioactivityandmetabolicclearanc e.Endocrinology.1995; 136(9):3839–3848), the biological activity and other properties of the recombinant CTP fusion protein are almost the same as those of the wild-type protein, but the half-life is relatively much longer; it proves that the recombinant CTP fusion The protein not only has good biological activity, but also has a long half-life in vivo.
目前延长蛋白半衰期的方法主要是利用PEG进行化学修饰，其优点是反应简单，能显著地增加被修饰蛋白质的体内半衰期；其缺点是修饰后蛋白分子量急剧增加，不利于体内代谢；化学修饰物多为异源物质，经常使用可能会引起人体的免疫应答；而且化学修饰后会使原有蛋白质的生物活性明显降低，一般仅为修饰前的10％；另外化学修饰的最终产物需要多进行一次纯化，得率相对较低，而且修饰剂本身价格不菲，生产成本相对较高。At present, the method of prolonging the half-life of protein is mainly to use PEG for chemical modification, which has the advantage of simple reaction and can significantly increase the in vivo half-life of the modified protein; the disadvantage is that the molecular weight of the modified protein increases sharply, which is not conducive to in vivo metabolism; there are many chemical modifications As a heterologous substance, frequent use may cause the body's immune response; and after chemical modification, the biological activity of the original protein will be significantly reduced, generally only 10% of that before modification; in addition, the final product of chemical modification needs to be purified once more , the yield is relatively low, and the modifier itself is expensive, and the production cost is relatively high.
基于上述现状，本申请的发明人拟提供一种长效的重组人粒细胞集落刺激因子的及其制备方法和用途，使制得的人粒细胞集落刺激因子的重组融合蛋白保持人粒细胞集落刺激因子的原有活性，延长该蛋白质在体内的半衰期，且免疫原性低。Based on the above situation, the inventors of the present application intend to provide a long-acting recombinant human granulocyte colony-stimulating factor and its preparation method and application, so that the recombinant fusion protein of human granulocyte colony-stimulating factor can maintain human granulocyte colonies Stimulates the original activity of the factor, prolongs the half-life of the protein in vivo, and has low immunogenicity.
发明内容Contents of the invention
本发明的目的是提供人粒细胞集落刺激因子的重组融合蛋白，具体涉及一种长效的重组人粒细胞集落刺激因子及其制备方法。本发明制得的重组融合蛋白在保持人粒细胞集落刺激因子的原有活性的前提下，能延长该蛋白质在体内的半衰期，且比同类化学修饰的长效化人粒细胞集落刺激因子产品具有更低的免疫原性。The purpose of the present invention is to provide a recombinant fusion protein of human granulocyte colony-stimulating factor, in particular to a long-acting recombinant human granulocyte colony-stimulating factor and a preparation method thereof. On the premise of maintaining the original activity of human granulocyte colony-stimulating factor, the recombinant fusion protein prepared by the present invention can prolong the half-life of the protein in vivo, and is more effective than similar chemically modified long-acting human granulocyte colony-stimulating factor products. Lower immunogenicity.
本发明的进一步目的是提供所述的重组融合蛋白在用于制备治疗放化疗或其他原因造成的中性粒细胞减少症以及骨髓抑制的药物中的用途。A further object of the present invention is to provide the use of the recombinant fusion protein in the preparation of drugs for treating neutropenia and bone marrow suppression caused by radiotherapy, chemotherapy or other reasons.
本发明要解决的关键技术问题是在延长重组人粒细胞集落刺激因子的体内半衰期的同时保持其体内外活性。The key technical problem to be solved by the present invention is to prolong the half-life of recombinant human granulocyte colony-stimulating factor in vivo and maintain its activity in vivo and in vitro.
本发明解决的技术方案是采用重组人粒细胞集落因子(rhG-CSF)和人绒毛膜促性腺激素(hCG)β亚基端118位到145位的氨基酸所组成的小肽(CTP)融合表达；该融合蛋白可在毕赤酵母中高效表达，所述制备工艺简单，可用于大规模的药物级融合蛋白的生产制备。The technical scheme solved by the present invention is the fusion expression of a small peptide (CTP) composed of recombinant human granulocyte colony factor (rhG-CSF) and human chorionic gonadotropin (hCG) β subbase terminal 118 to 145 amino acids ; The fusion protein can be efficiently expressed in Pichia pastoris, the preparation process is simple, and can be used for the production and preparation of large-scale pharmaceutical grade fusion protein.
具体的，本发明的一种长效的重组人粒细胞集落刺激因子，其特征在于，由一个重组人粒细胞集落刺激因子和一个或多个人绒毛膜促性腺激素(hCG)β亚基端118位到145位的氨基酸所组成的小肽(CTP)融合表达制成长效融合蛋白。Specifically, a long-acting recombinant human granulocyte colony-stimulating factor of the present invention is characterized in that it consists of a recombinant human granulocyte colony-stimulating factor and one or more human chorionic gonadotropin (hCG) beta subunit 118 A small peptide (CTP) composed of amino acids at position 145 is fused and expressed to make a long-acting fusion protein.
所述融合蛋白中人绒毛膜促性腺激素(hCG)β亚基端118位到145位的氨基酸所组成的小肽(CTP)的氨基酸序列为SEQIDNo.1所示。The amino acid sequence of the small peptide (CTP) composed of amino acids from the 118th to the 145th position of the beta subunit of human chorionic gonadotropin (hCG) in the fusion protein is shown in SEQ ID No.1.
SSSSKAPPPSLPSPSRLPGPSDTPILPQ(SEQIDNo.1)。SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID No. 1).
所述重组人粒细胞集落刺激因子(rhG-CSF)的氨基酸序列为SEQIDNo.2所示。The amino acid sequence of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is shown in SEQ ID No.2.
TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP(SEQIDNo.2)TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP (SEQ ID No. 2)
本发明在提供上述长效重组人粒细胞集落刺激因子融合蛋白的同时，提供了编码上述长效人粒细胞集落刺激因子融合蛋白的相关核苷酸序列。While providing the above-mentioned long-acting recombinant human granulocyte-colony-stimulating factor fusion protein, the present invention also provides related nucleotide sequences encoding the above-mentioned long-acting human granulocyte-colony-stimulating factor fusion protein.
本发明中，编码所述长效重组人粒细胞集落刺激因子融合蛋白中的人绒毛膜促性腺激素(hCG)β亚基端118位到145位的氨基酸所组成的小肽(CTP)的核苷酸序列如SEQIDNo.3所示：In the present invention, the nucleus encoding a small peptide (CTP) composed of amino acids from the 118th to 145th positions of the human chorionic gonadotropin (hCG) β subbase in the fusion protein of the long-acting recombinant human granulocyte colony-stimulating factor The nucleotide sequence is shown in SEQIDNo.3:
agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacag(SEQIDNo.3)agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacag (SEQ ID No. 3)
本发明中，所述重组人粒细胞集落刺激因子由一个重组人粒细胞集落刺激因子和一至多个CTP多肽融合表达制成；其组合方式包括但不限于：N-端的一个CTP和C-端的一个rhG-CSF融合(CTP-rhG-CSF)，N-端两个CTP和C-端的一个rhG-CSF融合(CTP-CTP-rhG-CSF)，N-端的rhG-CSF和C-端的一个CTP融合(rhG-CSF-CTP)，N-端的rhG-CSF和C-端的两个CTP融合，N-端的一个CTP及C-端的一个CTP共同与rhG-CSF融合(CTP-rhG-CSF-CTP)，或，N-端的一个CTP及C-端的两个CTP共同与rhG-CSF融合(CTP-rhG-CSF-CTP-CTP)。In the present invention, the recombinant human granulocyte colony-stimulating factor is produced by fusion expression of one recombinant human granulocyte colony-stimulating factor and one or more CTP polypeptides; the combination method includes but not limited to: a CTP at the N-terminal and a One rhG-CSF fusion (CTP-rhG-CSF), two CTPs at the N-terminus and one rhG-CSF fusion at the C-terminus (CTP-CTP-rhG-CSF), rhG-CSF at the N-terminus and one CTP at the C-terminus Fusion (rhG-CSF-CTP), N-terminal rhG-CSF and C-terminal two CTP fusion, N-terminal CTP and C-terminal CTP are fused together with rhG-CSF (CTP-rhG-CSF-CTP) , or, one CTP at the N-terminal and two CTPs at the C-terminal are fused together with rhG-CSF (CTP-rhG-CSF-CTP-CTP).
本发明中最优组合方式是N-端的一个CTP及C-端的两个CTP共同与rhG-CSF融合表达制成的融合蛋白CTP-rhG-CSF-CTP-CTP。The optimal combination in the present invention is a fusion protein CTP-rhG-CSF-CTP-CTP produced by fusion expression of one N-terminal CTP and two C-terminal CTPs together with rhG-CSF.
本发明中，编码所述长效重组人粒细胞集落刺激因子融合蛋白的核苷酸序列如SEQIDNo.4～9所示。In the present invention, the nucleotide sequence encoding the long-acting recombinant human granulocyte colony-stimulating factor fusion protein is shown in SEQ ID No. 4-9.
N-端的一个CTP和C-端的一个rhG-CSF融合(CTP-rhG-CSF)：One CTP at the N-terminus and one rhG-CSF at the C-terminus fused (CTP-rhG-CSF):
agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagacccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagccctga(SEQIDNo.4)agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagacccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagccctga(SEQIDNo.4)
N-端两个CTP和C-端的一个rhG-CSF融合(CTP-CTP-rhG-CSF)Fusion of two CTPs at the N-terminus and one rhG-CSF at the C-terminus (CTP-CTP-rhG-CSF)
agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagacccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagccctga(SEQIDNo.5)agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagacccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagccctga(SEQIDNo.5)
N-端的rhG-CSF和C-端的一个CTP融合(rhG-CSF-CTP)N-terminal rhG-CSF fused with a C-terminal CTP (rhG-CSF-CTP)
acccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagcccagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagtga(SEQIDNo.6)acccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagcccagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagtga(SEQIDNo.6)
N-端的rhG-CSF和C-端的两个CTP融合rhG-CSF at the N-terminus and two CTP fusions at the C-terminus
acccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggaggggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagcccagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagtga(SEQIDNo.7)acccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggaggggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagcccagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagtga(SEQIDNo.7)
N-端的一个CTP及C-端的一个CTP共同与rhG-CSF融合(CTP-rhG-CSF-CTP)：A CTP at the N-terminus and a CTP at the C-terminus are fused with rhG-CSF (CTP-rhG-CSF-CTP):
Agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagacccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagcccagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagtga(SEQIDNo.8)Agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagacccccctgggccctgccagctccctgccccagagcttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccaggagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacactctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggcaggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctggaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgactttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagcccacccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctagttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgcccagcccagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctgccacagtga(SEQIDNo.8)
N-端的一个CTP及C-端的两个CTP共同与rhG-CSF融合(CTP-rhG-CSF-CTP)：One CTP at the N-terminal and two CTPs at the C-terminal are fused with rhG-CSF (CTP-rhG-CSF-CTP):
AGTTCATCATCAAAAGCACCTCCTCCATCATTGCCATCCCCATCAAGATTGCCTGGTCCATCAGATACTCCTATATTGCCTCAAACACCTTTGGGTCCAGCATCTTCATTGCCACAATCATTTTTGTTGAAGTGTTTGGAACAAGTAAGAAAGATCCAAGGTGACGGTGCTGCATTGCAAGAAAAGTTGTGTGCTACTTACAAGTTGTGCCATCCTGAAGAATTAGTTTTGTTAGGTCACTCTTTGGGTATTCCTTGGGCACCATTATCCAGTTGTCCATCACAAGCTTTGCAATTAGCAGGTTGCTTGTCCCAATTGCATAGTGGTTTGTTTTTATACCAAGGTTTGTTACAAGCCTTAGAAGGTATTTCACCTGAATTGGGTCCAACCTTAGACACTTTGCAATTAGATGTTGCAGACTTCGCCACTACAATATGGCAACAAATGGAAGAATTGGGTATGGCCCCTGCTTTACAACCAACACAAGGTGCTATGCCTGCATTTGCCTCCGCTTTCCAAAGAAGAGCCGGTGGTGTTTTGGTAGCTTCTCATTTGCAATCATTCTTAGAAGTCTCTTACAGAGTTTTGAGACACTTAGCACAACCATCTTCATCCAGTAAAGCCCCACCTCCATCCTTGCCTTCTCCATCAAGATTACCTGGTCCAAGTGATACCCCTATATTACCACAATCTTCATCCAGTAAGGCTCCTCCACCTTCTTTACCATCCCCTTCCAGATTGCCAGGTCCTTCAGACACTCCTATTTTGCCACAATGA(SEQIDNo.9)AGTTCATCATCAAAAGCACCTCCTCCATCATTGCCATCCCCATCAAGATTGCCTGGTCCATCAGATACTCCTATATTGCCTCAAACACCTTTGGGTCCAGCATCTTCATTGCCACAATCATTTTTGTTGAAGTGTTTGGAACAAGTAAGAAAGATCCAAGGTGACGGTGCTGCATTGCAAGAAAAGTTGTGTGCTACTTACAAGTTGTGCCATCCTGAAGAATTAGTTTTGTTAGGTCACTCTTTGGGTATTCCTTGGGCACCATTATCCAGTTGTCCATCACAAGCTTTGCAATTAGCAGGTTGCTTGTCCCAATTGCATAGTGGTTTGTTTTTATACCAAGGTTTGTTACAAGCCTTAGAAGGTATTTCACCTGAATTGGGTCCAACCTTAGACACTTTGCAATTAGATGTTGCAGACTTCGCCACTACAATATGGCAACAAATGGAAGAATTGGGTATGGCCCCTGCTTTACAACCAACACAAGGTGCTATGCCTGCATTTGCCTCCGCTTTCCAAAGAAGAGCCGGTGGTGTTTTGGTAGCTTCTCATTTGCAATCATTCTTAGAAGTCTCTTACAGAGTTTTGAGACACTTAGCACAACCATCTTCATCCAGTAAAGCCCCACCTCCATCCTTGCCTTCTCCATCAAGATTACCTGGTCCAAGTGATACCCCTATATTACCACAATCTTCATCCAGTAAGGCTCCTCCACCTTCTTTACCATCCCCTTCCAGATTGCCAGGTCCTTCAGACACTCCTATTTTGCCACAATGA(SEQIDNo.9)
本发明还提供了含有所述重组人粒细胞集落刺激因子和所述CTP短肽的核苷酸序列的载体。The present invention also provides a vector containing the recombinant human granulocyte colony-stimulating factor and the nucleotide sequence of the CTP short peptide.
本发明还提供了含有所述重组人粒细胞集落刺激因子和所述CTP短肽的核苷酸序列的的宿主细胞。The present invention also provides host cells containing the recombinant human granulocyte colony-stimulating factor and the nucleotide sequence of the short CTP peptide.
本发明中，所述的宿主细胞包括但不限于大肠杆菌，毕赤酵母，和哺乳动物细胞；其中最优的是毕赤酵母。In the present invention, the host cells include but not limited to Escherichia coli, Pichia pastoris, and mammalian cells; the most preferred one is Pichia pastoris.
本发明的长效的重组人粒细胞集落刺激因子通过下述方法制备；The long-acting recombinant human granulocyte colony-stimulating factor of the present invention is prepared by the following method;
(1)构建重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白表达载体：(1) Construction of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein expression vector:
人工化学合成带酶切位点及信号肽的重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白编码基因序列，并将其克隆至pUC57simple质粒载体中，得到含有融合蛋白编码基因的质粒(由南京金斯瑞生物技术有限公司完成)；The recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein coding gene sequence with enzyme cleavage site and signal peptide was artificially synthesized, and cloned into the pUC57simple plasmid vector to obtain a plasmid containing the fusion protein coding gene (from Nanjing GenScript Biotechnology Co., Ltd. completed);
取pUC57simple-rhG-CSF-CTP3的质粒及pPICZaA表达质粒，采用XhoI及NotI限制性核酸内切酶进行双酶切,酶切产物采用1％琼脂糖凝胶电泳分离，采用胶回收试剂盒回收目的片段；Take the pUC57simple-rhG-CSF-CTP3 plasmid and the pPICZaA expression plasmid, and use XhoI and NotI restriction endonucleases for double digestion. The digested products are separated by 1% agarose gel electrophoresis, and the gel recovery kit is used for recovery. Purpose Fragment;
在T4DNA连接酶催化下进行连接反应，连接产物转化感受态大肠杆菌DH5α，涂布于Zeocin抗性LB平板培养，挑取单克隆，扩大培养后抽提质粒，采用NcoI及XhoI双酶切；The ligation reaction was carried out under the catalysis of T4DNA ligase, and the ligation product was transformed into competent Escherichia coli DH5α, spread on a Zeocin-resistant LB plate and cultured, picked a single clone, extracted the plasmid after expansion, and digested with NcoI and XhoI;
酶切产物采用1％琼脂糖凝胶电泳分离，观察酶切产物的大小，并测序；The digested product was separated by 1% agarose gel electrophoresis, the size of the digested product was observed, and sequenced;
(2)重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的表达(2) Expression of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein
制备感受态毕赤酵母GS115细胞，将测序正确的表达重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的表达载体pPICZaA-rhG-CSF-CTP3用限制性内切酶SacI进行单酶切线性化，并收集4000bp处的片段；然后将1线性化的转化pPICZaA-rhG-CSF-CTP3质粒在场强下转化感受态毕赤酵母GS115细胞，挑取单克隆转化子备用；Competent Pichia pastoris GS115 cells were prepared, and the expression vector pPICZaA-rhG-CSF-CTP3, which was correctly sequenced and expressed recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein, was single-digested with restriction endonuclease SacI and linearized. and collect the fragment at 4000bp; then transform the competent Pichia pastoris GS115 cells with 1 linearized transformed pPICZaA-rhG-CSF-CTP3 plasmid under the field strength, and pick the single clone transformants for later use;
将单克隆转化子转接入BMGY培养基连续培养后，取上清电泳检测结果显示，目的蛋白表达，大小与预期相符；After the monoclonal transformants were transferred to BMGY medium for continuous culture, the results of electrophoresis of the supernatant showed that the target protein was expressed and the size was in line with the expectation;
(3)重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的体外活性测定(3) Determination of in vitro activity of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein
选用G-CSF依赖细胞株NFS60,以MTT法测定生物学活性，结果如图3所示，rhG-CSF-CTP3的体外生物活性与G-CSF相似，显著高于同类PEG产品Neulasta。The G-CSF-dependent cell line NFS60 was selected, and the biological activity was determined by the MTT method. As shown in Figure 3, the in vitro biological activity of rhG-CSF-CTP3 was similar to that of G-CSF, and significantly higher than that of the similar PEG product Neulasta.
(4)重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的体内活性测定(4) Determination of in vivo activity of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein
采用环磷酰胺(CTX)建立小鼠白细胞低下模型测定融合蛋白体内活性结果显示，制得的融合蛋白rhG-CSF-CTP3与Neulasta及每天注射瑞白的药效程度相当，在注射7天后仍能保持较好的药效(如图4所示)。Cyclophosphamide (CTX) was used to establish a mouse leukopenia model to measure the in vivo activity of the fusion protein. The results showed that the fusion protein rhG-CSF-CTP3 was as effective as Neulasta and daily injection of Ruibai, and it could still be cured after 7 days of injection. Maintain better efficacy (as shown in Figure 4).
本发明进一步提供了上述长效人粒细胞集落刺激因子在用于制备治疗放化疗或其他原因造成的中性粒细胞减少症，骨髓抑制等的治疗药物中的用途。The present invention further provides the use of the above-mentioned long-acting human granulocyte colony-stimulating factor in the preparation of therapeutic drugs for treating neutropenia, bone marrow suppression, etc. caused by radiotherapy, chemotherapy or other reasons.
本发明的优点在于，The advantage of the present invention is that,
制得的长效的重组人粒细胞集落刺激因子不会引入任何异源物质，免疫原性相对于化学修饰后蛋白质更低；而且重组CTP融合蛋白为纯粹的基因工程表达产品，无需修饰剂，能较好地保持原有蛋白质的生物活性，并且产物得率相比化学修饰蛋白质有明显提高，使得整个生产成本大幅度降低。The prepared long-acting recombinant human granulocyte colony-stimulating factor will not introduce any heterologous substances, and its immunogenicity is lower than that of chemically modified proteins; and the recombinant CTP fusion protein is a purely genetic engineering expression product without modifiers, The biological activity of the original protein can be well maintained, and the product yield is significantly improved compared with the chemically modified protein, so that the entire production cost is greatly reduced.
附图说明Description of drawings
图1：重组表达载体的酶切鉴定。Figure 1: Enzyme digestion identification of recombinant expression vectors.
图2，重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白诱导表达的SDS-PAGE图谱，Figure 2, the SDS-PAGE map of the induced expression of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein,
其中，1，甲醇诱导组；2，甲醇诱导组；3，对照组，4，对照组。Among them, 1, methanol induction group; 2, methanol induction group; 3, control group, 4, control group.
图3，重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的体外活性测定。Fig. 3, the in vitro activity determination of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein.
图4，重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的体内活性测定。Fig. 4, the in vivo activity determination of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein.
具体实施方式detailed description
实施例1：重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白表达载体的构建：Example 1: Construction of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein expression vector:
人工化学合成带酶切位点及信号肽的重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白编码基因序列，并将其克隆至pUC57simple质粒载体中，得到含有融合蛋白编码基因的质粒。(由南京金斯瑞生物技术有限公司完成)。该质粒融合蛋白的5’端含有XhoI酶切位点和信号肽剪切识别位点(即5’CTCGAGAAAAGA-)；且3’端带有NotI酶切位点(即3’GCGGCCGC-)The recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein coding gene sequence with enzyme cleavage site and signal peptide was artificially synthesized, and cloned into the pUC57simple plasmid vector to obtain a plasmid containing the fusion protein coding gene. (Completed by Nanjing GenScript Biotechnology Co., Ltd.). The 5' end of the plasmid fusion protein contains an XhoI restriction site and a signal peptide cleavage recognition site (i.e. 5'CTCGAGAAAAAGA-); and a NotI restriction site at the 3' end (i.e. 3'GCGGCCGC-)
取pUC57simple-rhG-CSF-CTP3的质粒及pPICZaA表达质粒，采用XhoI及NotI限制性核酸内切酶进行双酶切。双酶切的反应体系为20μL，其中质粒10μL，NotI1μL，XhoI1μL，10XbufferH2μL,无菌水6μL。37℃酶切5h。酶切产物采用1％琼脂糖凝胶电泳分离，、采用胶回收试剂盒回收目的片段。其中pPiczaA表达载体回收3500bp左右的片段，pUC57simple-rhG-CSF-CTP3载体回收750bp的片段。Take the pUC57simple-rhG-CSF-CTP3 plasmid and the pPICZaA expression plasmid, and use XhoI and NotI restriction endonucleases for double digestion. The reaction system for double enzyme digestion is 20 μL, including 10 μL of plasmid, 1 μL of NotI, 1 μL of XhoI, 2 μL of 10XbufferH, and 6 μL of sterile water. Enzyme digestion at 37°C for 5h. The digested product was separated by 1% agarose gel electrophoresis, and the target fragment was recovered using a gel extraction kit. Among them, a fragment of about 3500 bp was recovered from the pPiczaA expression vector, and a fragment of 750 bp was recovered from the pUC57simple-rhG-CSF-CTP3 vector.
连接反应在T4DNA连接酶催化下进行，pPiczaA表达载体酶切片段与pUC57simple-rhG-CSF-CTP3载体的摩尔数比约为1:4。连接反应体系为25μL，其中T4连接酶1μL，10XT4DNA连接酶缓冲液2.5μL，16℃连接过夜。The ligation reaction was carried out under the catalysis of T4DNA ligase, and the molar ratio of pPiczaA expression vector digestion fragment to pUC57simple-rhG-CSF-CTP3 vector was about 1:4. The ligation reaction system was 25 μL, including 1 μL of T4 ligase, 2.5 μL of 10XT4 DNA ligase buffer, and ligated overnight at 16°C.
连接产物转化感受态大肠杆菌DH5α，涂布于Zeocin抗性LB平板上，37℃培养过夜，挑取单克隆，扩大培养后抽提质粒，采用NcoI及XhoI双酶切。酶切产物采用1％琼脂糖凝胶电泳分离，观察酶切产物的大小，实验结果如图1所示。将扩大培养的菌液采用pET载体通用引物测序，测序反应由上海英潍捷基贸易有限公司完成；经测定，双酶切产物大小约为750bp，与设计的792bp基本相同，且测序结果显示设计的rhG-CSF-CTP3的序列和实际构建的融合蛋白的核苷酸序列完全相同。The ligation product was transformed into competent Escherichia coli DH5α, spread on a Zeocin-resistant LB plate, cultured overnight at 37°C, picked a single clone, extracted the plasmid after expansion, and digested with NcoI and XhoI. The digested product was separated by 1% agarose gel electrophoresis, and the size of the digested product was observed. The experimental results are shown in FIG. 1 . The expanded cultured bacterial liquid was sequenced with pET vector universal primers, and the sequencing reaction was completed by Shanghai Yingwei Jieji Trading Co., Ltd.; after measurement, the size of the double enzyme digestion product was about 750bp, which was basically the same as the designed 792bp, and the sequencing results showed that the designed The rhG-CSF-CTP3 sequence is completely identical to the nucleotide sequence of the actually constructed fusion protein.
实施例2重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的表达Example 2 Expression of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein
首先制备感受态毕赤酵母GS115细胞，具体步骤如下：首先挑取GS115的单克隆菌落于3mL的YPD培养基中，以180rpm的转速于30℃恒温摇动孵育48h。然后以1:200的接种量将培养好的毕赤酵母GS115细胞转接入100mL的YPD培养基中，培养至O.D.600＝1.2。以4000rpm/min4℃离心5分钟，弃上清，收集沉淀以1mol/L的山梨醇溶液重悬两次后，以4000rpm/min4℃离心10分钟，用100μL的冰冷山梨醇溶液重悬沉淀，即制成感受态毕赤酵母GS115细胞。Competent Pichia pastoris GS115 cells were prepared first, and the specific steps were as follows: First, single-clonal colonies of GS115 were picked and placed in 3 mL of YPD medium, and incubated at 30° C. for 48 hours at a speed of 180 rpm. Then transfer the cultured Pichia pastoris GS115 cells into 100 mL of YPD medium at an inoculum size of 1:200, and culture to O.D.600=1.2. Centrifuge at 4000rpm/min at 4°C for 5 minutes, discard the supernatant, collect the precipitate and resuspend twice with 1mol/L sorbitol solution, centrifuge at 4000rpm/min at 4°C for 10 minutes, and resuspend the precipitate with 100μL of ice-cold sorbitol solution, that is Competent Pichia pastoris GS115 cells were made.
其次将测序正确的表达重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的表达载体pPICZaA-rhG-CSF-CTP3用限制性内切酶SacI进行单酶切线性化，并收集4000bp处的片段。Secondly, the expression vector pPICZaA-rhG-CSF-CTP3 expressing the fusion protein of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP correctly sequenced was linearized with restriction endonuclease SacI, and the fragment at 4000bp was collected .
然后将10μg线性化好的转化pPICZaA-rhG-CSF-CTP3质粒在1000mV/mm的场强下转化感受态毕赤酵母GS115细胞。活化感受态细胞并涂在含有Zeocin的抗性板上培养。挑取单克隆转化子备用。Then, 10 μg of the linearized transformed pPICZaA-rhG-CSF-CTP3 plasmid was transformed into competent Pichia pastoris GS115 cells at a field strength of 1000 mV/mm. Competent cells were activated and plated on Zeocin-containing resistant plates for culture. Single clone transformants were picked for use.
将单克隆转化子转接入5mLBMGY培养基中于30℃恒温连续振荡培养48h后离心5min弃上清，加入5mLBMMY培养基，并每日往培养基中加入0.5％的甲醇诱导表达。连续恒温连续振荡培养96h。收集培养液上清进行SDS-PAGE电泳检测。结果如图2所示。结果表明与未经甲醇诱导对照组相比，甲醇诱导的实验组在45KD附近有目的蛋白表达，大小与预期相符。The monoclonal transformant was transferred into 5mL BMGY medium, cultured at 30°C with constant temperature and continuous shaking for 48h, centrifuged for 5min, discarded the supernatant, added 5mL BMMY medium, and added 0.5% methanol to the medium every day to induce expression. Continuous constant temperature and continuous shaking culture for 96h. The culture supernatant was collected for SDS-PAGE electrophoresis detection. The result is shown in Figure 2. The results showed that compared with the control group without methanol induction, the experimental group induced by methanol expressed the target protein around 45KD, and the size was consistent with the expectation.
实施例3重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的体外活性测定Example 3 Determination of In Vitro Activity of Recombinant Human Granulocyte Colony Stimulating Factor rhG-CSF and CTP Fusion Protein
本实施例依照中华人民共和国药典(2010版，三部)，选用G-CSF依赖细胞株NFS60,以MTT法测定生物学活性。具体步骤如下：FS60细胞株用完全培养液于37℃、5％CO2培养，控制细胞浓度为每毫升含1*105个细胞用于测定。取足量NFS60细胞培养物，离心收集细胞，用RPMI1640培养液洗两次，然后重悬于基础培养液(RPMI1640900ml+新生牛血清100ml)配成每毫升含2*105个细胞的细胞悬液。在加有标准品和供试品的96孔板中每孔加入细胞悬液50μL，于37℃、5％CO2培养72小时。每孔加入MTT溶液20μL，于37℃、5％CO2培养5小时。每孔加入裂解液100μL，37℃过夜，用酶标仪于波长570nm处测定吸光度。结果如图3所示，rhG-CSF-CTP3的体外生物活性与G-CSF相似，显著高于同类PEG产品Neulasta。In this example, according to the Pharmacopoeia of the People's Republic of China (2010 Edition, Volume III), the G-CSF-dependent cell line NFS60 was selected, and the biological activity was determined by the MTT method. The specific steps are as follows: FS60 cell line was cultured with complete culture medium at 37° C. and 5% CO2 , and the cell concentration was controlled to contain 1*105 cells per milliliter for determination. Take a sufficient amount of NFS60 cell culture, collect the cells by centrifugation, wash twice with RPMI1640 culture medium, and then resuspend in basal culture medium (RPMI1640900ml + newborn bovine serum 100ml) to make a cell suspension containing2 *105 cells per ml. Add 50 μL of cell suspension to each well of a 96-well plate with standard and test products, and incubate at 37° C., 5% CO2 for 72 hours. Add 20 μL of MTT solution to each well, and incubate at 37° C., 5% CO2 for 5 hours. Add 100 μL of lysate to each well, overnight at 37°C, and measure the absorbance at a wavelength of 570 nm with a microplate reader. The results are shown in Figure 3, the in vitro biological activity of rhG-CSF-CTP3 is similar to that of G-CSF, and significantly higher than that of the similar PEG product Neulasta.
实施例4重组人粒细胞集落刺激因子rhG-CSF与CTP融合蛋白的体内活性测定Example 4 In vivo activity determination of recombinant human granulocyte colony-stimulating factor rhG-CSF and CTP fusion protein
用环磷酰胺(CTX)建立小鼠白细胞低下模型测定融合蛋白体内活性，以200mg/kg的剂量注射于BALB/c小鼠皮下，测定3天小鼠白细胞数，发现小鼠的白细胞数连续3天均低于5000个/mm3.所有建模小鼠从第四天开始给药，实验组于实验开始第一天的尾静脉注射一次rhG-CSF-CTP3融合蛋白，共计注射一次。阳性对照组分别注射Neulasta和瑞白(rh-GCSF)。而阴性对照组则注射醋酸缓冲液。连续观察12天。每天取小鼠尾部末梢静脉血计数白细胞的数量，统计结果如图4所示，在12天内，融合蛋白rhG-CSF-CTP3与Neulasta及每天注射瑞白的药效程度相当，在注射7天后仍能保持较好的药效。Cyclophosphamide (CTX) was used to establish a mouse leukopenia model to measure the in vivo activity of the fusion protein, inject it subcutaneously in BALB/c mice at a dose of 200 mg/kg, and measure the number of white blood cells in the mice for 3 days. It was found that the number of white blood cells in the mice continued for 3 The daily average was less than 5000 cells/mm3 . All model mice were administered from the fourth day, and the experimental group was injected with rhG-CSF-CTP3 fusion protein once in the tail vein on the first day of the experiment, for a total of one injection. The positive control group was injected with Neulasta and Ruibai (rh-GCSF). The negative control group was injected with acetate buffer. Continuous observation for 12 days. The number of white blood cells was counted in the peripheral vein blood of the tail of the mouse every day. The statistical results are shown in Figure 4. Within 12 days, the fusion protein rhG-CSF-CTP3 had the same drug effect as Neulasta and the daily injection of Ruibai. Can maintain better efficacy.
SEQUENCELISTINGSEQUENCELISTING
<110>复旦大学<110> Fudan University
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cctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctg600cctccaccttccctgcccagtccaagccgactccctgggccctccgatacaccaattctg600
ccacagagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccct660ccacagagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccct660
gggccctccgatacaccaattctgccacagtga693gggccctccgatacaccaattctgccacagtga693
<210>8<210>8
<211>692<211>692
<212>DNA<212>DNA
<213>Artificial<213>Artificial
<400>8<400>8
agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccc60agctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctgggccc60
tccgatacaccaattctgccacagacccccctgggccctgccagctccctgccccagagc120tccgatacaccaattctgccacagaccccccctgggccctgccagctccctgccccagagc120
ttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccag180ttcctgctcaagtgcttagagcaagtgaggaagatccagggcgatggcgcagcgctccag180
gagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacac240gagaagctgtgtgccacctacaagctgtgccaccccgaggagctggtgctgctcggacac240
tctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggca300tctctgggcatcccctgggctcccctgagcagctgccccagccaggccctgcagctggca300
ggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctg360ggctgcttgagccaactccatagcggccttttcctctaccaggggctcctgcaggccctg360
gaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgac420gaagggatctcccccgagttgggtcccaccttggacacactgcagctggacgtcgccgac420
tttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagccc480tttgccaccaccatctggcagcagatggaagaactgggaatggcccctgccctgcagccc480
acccagggtgccatgccggccttcgcctctgctttccagcgccgggcaggagggtcctag540acccagggtgccatgccggccttcgcctctgctttccagcgccggggcaggagggtcctag540
ttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgccc600ttgcctcccatctgcagagcttcctggaggtgtcgtaccgcgttctacgccaccttgccc600
agcccagctccagcagcaaggcccctccaccttccctgcccagtccaagccgactccctg660agcccagctccagcagcaaggcccctccaccttccctgccccagtccaagccgactccctg660
ggccctccgatacaccaattctgccacagtga692ggccctccgatacaccaattctgccacagtga692
<210>9<210>9
<211>777<211>777
<212>DNA<212>DNA
<213>Artificial<213>Artificial
<400>9<400>9
agttcatcatcaaaagcacctcctccatcattgccatccccatcaagattgcctggtcca60agttcatcatcaaaagcacctcctccatcattgccatccccatcaagattgcctggtcca60
tcagatactcctatattgcctcaaacacctttgggtccagcatcttcattgccacaatca120tcagatactcctatattgcctcaaacacctttgggtccagcatcttcattgccacaatca120
tttttgttgaagtgtttggaacaagtaagaaagatccaaggtgacggtgctgcattgcaa180tttttgttgaagtgtttggaacaagtaagaaagatccaaggtgacggtgctgcattgcaa180
gaaaagttgtgtgctacttacaagttgtgccatcctgaagaattagttttgttaggtcac240gaaaagttgtgtgctacttacaagttgtgccatcctgaagaattagttttgttaggtcac240
tctttgggtattccttgggcaccattatccagttgtccatcacaagctttgcaattagca300tctttgggtattccttgggcaccattatccagttgtccatcacaagctttgcaattagca300
ggttgcttgtcccaattgcatagtggtttgtttttataccaaggtttgttacaagcctta360ggttgcttgtcccaattgcatagtggtttgtttttaccaaggtttgttacaagcctta360
gaaggtatttcacctgaattgggtccaaccttagacactttgcaattagatgttgcagac420gaaggtatttcacctgaattgggtccaaccttagacactttgcaattagatgttgcagac420
ttcgccactacaatatggcaacaaatggaagaattgggtatggcccctgctttacaacca480ttcgccactacaatatggcaacaaatggaagaattgggtatggcccctgctttacaacca480
acacaaggtgctatgcctgcatttgcctccgctttccaaagaagagccggtggtgttttg540acacaaggtgctatgcctgcatttgcctccgctttccaaagaagagccggtggtgttttg540
gtagcttctcatttgcaatcattcttagaagtctcttacagagttttgagacacttagca600gtagcttctcatttgcaatcattcttagaagtctcttacagagttttgagacacttagca600
caaccatcttcatccagtaaagccccacctccatccttgccttctccatcaagattacct660caaccatcttcatccagtaaagccccacctccatccttgccttctccatcaagattacct660
ggtccaagtgatacccctatattaccacaatcttcatccagtaaggctcctccaccttct720ggtccaagtgatacccctatattaccacaatcttcatccagtaaggctcctccaccttct720
ttaccatccccttccagattgccaggtccttcagacactcctattttgccacaatga777ttaccatccccttccagattgccaggtccttcagacactcctattttgccacaatga777
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410439810.7ACN105461810A (en) | 2014-08-31 | 2014-08-31 | Long-acting recombinant human granulocyte colony-stimulating factor and preparation method therefor and use thereof |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410439810.7ACN105461810A (en) | 2014-08-31 | 2014-08-31 | Long-acting recombinant human granulocyte colony-stimulating factor and preparation method therefor and use thereof |
| Publication Number | Publication Date |
|---|---|
| CN105461810Atrue CN105461810A (en) | 2016-04-06 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410439810.7APendingCN105461810A (en) | 2014-08-31 | 2014-08-31 | Long-acting recombinant human granulocyte colony-stimulating factor and preparation method therefor and use thereof |
| Country | Link |
|---|---|
| CN (1) | CN105461810A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021078834A1 (en)* | 2019-10-22 | 2021-04-29 | Genethon | Chimeric acid-alpha glucosidase polypeptides and uses thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405182A (en)* | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and granulocyte colony stimulating factor fusion protein |
| US8048849B2 (en)* | 2006-02-03 | 2011-11-01 | Modigene, Inc. | Long-acting polypeptides and methods of producing same |
| WO2014080401A2 (en)* | 2012-11-20 | 2014-05-30 | Prolor Biotech Inc | Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405182A (en)* | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and granulocyte colony stimulating factor fusion protein |
| US8048849B2 (en)* | 2006-02-03 | 2011-11-01 | Modigene, Inc. | Long-acting polypeptides and methods of producing same |
| WO2014080401A2 (en)* | 2012-11-20 | 2014-05-30 | Prolor Biotech Inc | Method of increasing the hydrodynamic volume of polypeptides by attaching to gonadotrophin carboxy terminal peptides |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021078834A1 (en)* | 2019-10-22 | 2021-04-29 | Genethon | Chimeric acid-alpha glucosidase polypeptides and uses thereof |
| Publication | Publication Date | Title |
|---|---|---|
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