重组免疫毒素CTLA4-ScFv-Mel的构建、制备及活性研究

发布时间：2018-05-14 10:16

本文选题：重组免疫毒素 + CTLA-4　；参考：《中国人民解放军军医进修学院》2012年博士论文

【摘要】：器官移植是治疗终末期器官功能衰竭的有效手段,免疫抑制剂的应用可以减轻移植排斥反应,但化学类免疫抑制剂毒副作用明显,影响了其临床应用。生物制剂类免疫抑制剂尤其是免疫毒素可以通过其特异性的结合作用,清除抗原特异性T细胞,且毒副作用较小,有更好的应用前景。免疫毒素是具有导向能力的分子(载体)和具有细胞毒性的分子(毒素)偶联而成的具有特异性细胞杀伤能力的杂合分子,在治疗恶性肿瘤、移植排斥等方面有良好的应用前景。 CTLA-4是一种仅表达于活化T细胞而静止T细胞不表达的分子,因此CTLA-4的抗原结合分子是一种理想的免疫毒素导向分子,由之产生的免疫毒素只对活化的T细胞或肿瘤细胞有杀伤作用,避免了对正常静息T细胞的非特异性损伤。蜂毒肽(Melittin, Mel)是蜜蜂蜂毒的主要成分,是一种重要的抗菌肽,具有抗菌、抗辐射、抗肿瘤等作用。它由26个氨基酸组成,分子量小,免疫原性低,不易产生过敏反应；具有膜活性,直接对细胞的磷脂膜起溶解作用,抑制细胞发育,是一种较理想毒素片段。本研究以人源化抗CTLA-4单链抗体为靶向片段,特异性结合在表达CTLA-4的活化T细胞表面,以蜂毒肽类似物为毒素片段,特异性杀伤活化T细胞,设计构建出了高效低毒的免疫毒素分子,主要用于抗移植排斥反应。利用重叠延伸PCR技术,通过两步PCR获得了重组免疫毒素全长基因,将其与表达载体pBV220连接,转化大肠杆菌,重组基因在大肠杆菌表达系统中以包涵体形式表达。包涵体用8M脲变性,采用稀释复性,然后用SP-Sepharose-Fast-Flow阳离子交换柱分离纯化,Sephacryl S-200凝胶排阻层析柱精制,制备得到目的蛋白CTLA4-ScFv-Mel. 在体外活性研究中,2μmo1/L的CTLA4-ScFv-Mel分别作用于非活化的T淋巴细胞、阴性对照细胞ECV-304、ConA激活的T淋巴细胞、CTLA-4阳性细胞Raji知Jurkat细胞。结果显示,给药24h后各组细胞存活率下降,其中前两组细胞的存活率分别为78.9%和77.3%；而后三组分别为31.2%、32.7%和30.8%。说明CTLA4-ScFv-Mel对CTLA-4阳性细胞的杀伤率高于正常细胞,表现出细胞杀伤的选择性和高效率。体内活性研究以BEL-7402肝癌移植瘤模型和S180小鼠移植瘤模型为对象,分低、中、高三个剂量给药,比较试验组和阴性对照组移植瘤相对体积等药效学指标。结果显示,CTLA4-ScFv-Mel能够发挥促进两个试验组模型中移植瘤的存活和生长的作用,且呈现一定的剂量效应关系。推测该现象的出现与抑制T细胞免疫有关。本研究成功制备了一种全新的免疫毒素融合蛋白,并进行了初步活性研究和药效学评价,为器官移植提供了潜在的选择药物,具有一定的科学价值和开发前景。
[Abstract]:Organ transplantation is an effective method for the treatment of end-stage organ failure. The application of immunosuppressant can alleviate the rejection of transplantation, but the toxicity and side effect of chemical immunosuppressant is obvious, which affects its clinical application. The immunosuppressants, especially the immunotoxins, can remove antigen-specific T cells through their specific binding, and the toxicity and side effects are relatively small, so it has a better application prospect. Immunotoxins are conjugated molecules (vectors) that have the ability to direct and have cytotoxic molecules (toxins) to form hybrids with specific cytotoxic abilities in the treatment of malignant tumors. Transplantation rejection and other aspects have a good application prospects. CTLA-4 is a molecule expressed only in activated T cells but not expressed in static T cells. Therefore, antigen-binding molecules of CTLA-4 are ideal immunotoxin oriented molecules. The resulting immunotoxins can only kill activated T cells or tumor cells and avoid nonspecific damage to normal resting T cells. Melittin (melittin) is the main component of bee venom and is an important antimicrobial peptide. It has antibacterial, anti-radiation and anti-tumor effects. It is composed of 26 amino acids with low molecular weight and low immunogenicity, which is not easy to produce allergic reaction. It has membrane activity, which directly dissolves phospholipid membrane and inhibits cell development. It is an ideal toxin fragment. In this study, the humanized single chain antibody against CTLA-4 was used as the target fragment, specifically binding to the surface of activated T cells expressing CTLA-4, and the propolis analogue was used as the toxin fragment to specifically kill activated T cells. A highly effective and low-toxic immunotoxin molecule was designed and constructed, which was mainly used to resist transplant rejection. The full-length gene of recombinant immunotoxin was obtained by two-step PCR using overlapping extension PCR technique. The recombinant gene was ligated with the expression vector pBV220 and transformed into E. coli. The recombinant gene was expressed in the form of inclusion body in the expression system of Escherichia coli. The inclusion bodies were denatured with 8M-urea, then refolded by dilution, then purified by SP-Sepharose-Fast-Flow cationic exchange column, then purified by Sephacryl S-200 gel exclusion chromatography. The target protein CTLA4-ScFv-Melwas obtained. In vitro activity study, 2 渭 mo1/L CTLA4-ScFv-Mel acted on non-activated T lymphocytes, while negative control cells, ECV-304 Cona activated T lymphocytes, Raji positive cells known Jurkat cells. The results showed that the cell survival rate of the first two groups was 78.9% and 77.3%, respectively, while that of the latter three groups was 31.2% and 30.8%, respectively. The results showed that the killing rate of CTLA4-ScFv-Mel to CTLA-4 positive cells was higher than that of normal cells, which showed the selectivity and high efficiency of cell killing. In vivo activity study was carried out on BEL-7402 liver cancer transplanted tumor model and S180 mouse transplanted tumor model. The pharmacodynamic indexes were compared between the experimental group and the negative control group at three doses: low, middle and high doses. The results showed that CTLA4-ScFv-Mel could promote the survival and growth of transplanted tumor in two experimental groups, and showed a dose-effect relationship. It is speculated that the occurrence of this phenomenon is related to the suppression of T cell immunity. In this study, a novel fusion protein of immunotoxin was successfully prepared, and the preliminary activity and pharmacodynamics evaluation were carried out, which provided a potential drug selection for organ transplantation, which has certain scientific value and development prospect.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.4

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