抗CD3人鼠嵌合抗体的克隆及其在毕赤酵母中的表达
发布时间：2018-12-26 10:30
【摘要】： 自1986年美国FDA正式批准抗CD3单抗作为临床上治疗肾移植排斥反应的新药上市以来,抗CD3单抗以其具有激活和抑制人T细胞双向功能的特点,在各种器官移植排斥反应、肿瘤及自身免疫性疾病治疗中显示出其广阔的应用前景。但目前临床上应用的CD3单抗主要是鼠源性的,鼠源单抗在人体内应用时产生排斥反应、半衰期短、活性下降、异性蛋白反应等问题,严重限制其在临床治疗中的广泛应用。因此,近年来对于人源性的抗CD3抗体的研究成为热点。大多数抗CD3抗体基因构建后主要选用大肠杆菌和哺乳动物细胞为表达体系,这些表达体系都存在许多缺点。 酵母表达体系具备易于操作,遗传背景清楚,生产成本低等原核生物的特点,又具有真核生物的折叠、分泌机制,可以完成真核生物特异的蛋白酶解、折叠、糖基化等翻译后修饰过程,且生长速度快,培养成分简单,能适应工业化生产的需要。本研究选择酵母偏爱密码子同义替换鼠源性抗CD3重链可变区基因(Vh),与人源性重链恒定区(Ch)基因连接,构建完整的人鼠嵌合抗体。将构建的完整的人鼠嵌合表达载体转化X-33毕赤酵母,建立兼具原核系统良好操作性和真核系统后加工性的重组抗CD3毕赤酵母真核表达体系。通过ELISA和MTT的鉴定,筛选出高表达重组抗CD3的酵母菌株。结果表明,本研究正确构建了重组抗CD3抗体毕赤酵母真核表达体系,经甲醇诱导能够分泌具有活性的重组抗CD3抗体蛋白,相对分子量为55kD;重组抗CD3抗体的表达具有累积效应,产量在一段时间内随表达时间延长而增加。本研究为进一步用毕赤酵母作为生物反应器大规模生产抗CD3抗体提供了实验依据。
[Abstract]:Since the United States FDA officially approved anti-CD3 monoclonal antibody as a new drug for the treatment of renal transplantation rejection in 1986, anti-CD3 monoclonal antibody has been used in various organ transplantation rejection because of its bidirectional function of activating and inhibiting human T cells. The treatment of tumor and autoimmune disease shows its broad application prospect. However, the current clinical use of CD3 monoclonal antibody is mainly murine origin, the mouse source monoclonal antibody in human body produced rejection, short half-life, decreased activity, heterosexual protein reaction and other problems, which seriously limits its wide application in clinical treatment. Therefore, the study of human anti-CD3 antibody has become a hot topic in recent years. Escherichia coli and mammalian cells were used as expression systems after construction of anti CD3 antibody genes, and these expression systems had many disadvantages. The yeast expression system is characterized by easy operation, clear genetic background, low production cost and the mechanism of eukaryote folding and secretion, which can complete the proteolysis and folding of eukaryote. Glycosylation and other post-translational modification processes, with rapid growth, simple culture ingredients, can meet the needs of industrial production. In this study, yeast preference codon synonymous substitution of mouse anti CD3 heavy chain variable region gene (Vh), and human origin heavy chain constant region (Ch) gene was selected to construct a complete human mouse chimeric antibody. The constructed human mouse chimeric expression vector was transformed into Pichia pastoris X-33, and a recombinant eukaryotic expression system of anti-Pichia pastoris CD3 was established with good prokaryotic system operation and post-processing of eukaryotic system. Through the identification of ELISA and MTT, yeast strains with high expression of recombinant CD3 resistant to CD3 were screened out. The results showed that the eukaryotic expression system of recombinant anti CD3 antibody Pichia pastoris was constructed correctly. The recombinant anti CD3 antibody protein was secreted by methanol and its relative molecular weight was 55 kD. The expression of recombinant anti-CD3 antibody had cumulative effect, and the yield increased with the time of expression. This study provides experimental basis for mass production of anti-CD3 antibodies using Pichia pastoris as a bioreactor.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：Q786;R392
本文编号：2391965
[Abstract]:Since the United States FDA officially approved anti-CD3 monoclonal antibody as a new drug for the treatment of renal transplantation rejection in 1986, anti-CD3 monoclonal antibody has been used in various organ transplantation rejection because of its bidirectional function of activating and inhibiting human T cells. The treatment of tumor and autoimmune disease shows its broad application prospect. However, the current clinical use of CD3 monoclonal antibody is mainly murine origin, the mouse source monoclonal antibody in human body produced rejection, short half-life, decreased activity, heterosexual protein reaction and other problems, which seriously limits its wide application in clinical treatment. Therefore, the study of human anti-CD3 antibody has become a hot topic in recent years. Escherichia coli and mammalian cells were used as expression systems after construction of anti CD3 antibody genes, and these expression systems had many disadvantages. The yeast expression system is characterized by easy operation, clear genetic background, low production cost and the mechanism of eukaryote folding and secretion, which can complete the proteolysis and folding of eukaryote. Glycosylation and other post-translational modification processes, with rapid growth, simple culture ingredients, can meet the needs of industrial production. In this study, yeast preference codon synonymous substitution of mouse anti CD3 heavy chain variable region gene (Vh), and human origin heavy chain constant region (Ch) gene was selected to construct a complete human mouse chimeric antibody. The constructed human mouse chimeric expression vector was transformed into Pichia pastoris X-33, and a recombinant eukaryotic expression system of anti-Pichia pastoris CD3 was established with good prokaryotic system operation and post-processing of eukaryotic system. Through the identification of ELISA and MTT, yeast strains with high expression of recombinant CD3 resistant to CD3 were screened out. The results showed that the eukaryotic expression system of recombinant anti CD3 antibody Pichia pastoris was constructed correctly. The recombinant anti CD3 antibody protein was secreted by methanol and its relative molecular weight was 55 kD. The expression of recombinant anti-CD3 antibody had cumulative effect, and the yield increased with the time of expression. This study provides experimental basis for mass production of anti-CD3 antibodies using Pichia pastoris as a bioreactor.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：Q786;R392
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相关期刊论文 前3条
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