一种鼠源抗CTLA4单链抗体mS的构建及制备

发布时间：2018-01-09 04:09

  本文关键词：一种鼠源抗CTLA4单链抗体mS的构建及制备　出处：《四川大学》2006年硕士论文　论文类型：学位论文

  更多相关文章： CTLA-4 scFv 包涵体 复性 ELISA

【摘要】：免疫抑制剂在临床上的广泛应用是二十世纪器官移植领域取得的重大突破性进展之一。免疫抑制剂的广泛应用在大规模降低急性排斥反应发生率的同时，也引起了长期蓄积的毒副作用，从而导致移植器官的损伤，制约了病人或供器官长期存活率的进一步提高。已有研究表明，活化T细胞是介导移植排斥的重要细胞，选择性清除活化T细胞而保留静息T细胞，能控制移植排斥反应发生，减少药物的毒副作用。 活化的T细胞表面会诱导表达细胞毒性T淋巴细胞相关抗原4(CTLA4)分子，，基于识别CTLA4分子而杀伤活化T细胞的免疫毒素可以特异性杀伤活化T细胞而保留静息T细胞。作为免疫毒素关键的导向分子决定着目标分子的识别，详细探讨导向分子的功能对构建免疫毒素非常必要。基于此，我们设计鼠源anti-CTLA-4单链抗体(scFv)(简称mS)，用大肠杆菌表达系统表达该单链抗体，初步探索其功能，结果如下： 以装载了鼠源Anti-CTLA-4-scFv基因的PBMN质粒为模板，根据鼠源Anti-CTLA-4-scFv的基因序列设计引物，在引物设计时引入相关双酶切位点(BamH Ⅰ／Kpn Ⅰ)，PCR扩增获得单链抗体基因，再克隆到pQE30载体，提取pQE30-mS质粒，转入大肠杆菌中表达，提取表达产物发现蛋白mS绝大部分以包涵体形式表达，占细菌总蛋白量30％，极少量以
[Abstract]:The wide application of immunosuppressants in clinical practice is one of the most important breakthroughs in organ transplantation in 20th century. The extensive application of immunosuppressants can reduce the incidence of acute rejection on a large scale. It also causes long-term accumulation of toxic side effects, resulting in organ transplantation damage, restricting the patient or donor organ long-term survival rate of further improvement. Activated T cells are important cells in mediating transplantation rejection. Selective clearance of activated T cells and retention of resting T cells can control the occurrence of transplant rejection and reduce the toxic and side effects of drugs. Activated T cells can induce the expression of cytotoxic T lymphocyte associated antigen (CTLA4) molecules. Immunotoxins that kill activated T cells based on the recognition of CTLA4 molecules can specifically kill activated T cells and retain resting T cells. As the key guiding molecules of immunotoxins, the recognition of target molecules is determined. A detailed study of the function of the directed molecules is necessary for the construction of immunotoxins. Based on this, we designed murine anti-CTLA-4 scFvv (short for mSs). The scFv was expressed in E. coli expression system and its function was preliminarily explored. The results are as follows: Using the PBMN plasmid loaded with mouse Anti-CTLA-4-scFv gene as template, primers were designed according to the gene sequence of mouse Anti-CTLA-4-scFv. The single chain antibody (scFv) gene was amplified by polymerase chain reaction with BamH 鈪

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