SrTRAIL的表达与DR5单抗的人源化改造

发布时间：2018-05-28 18:56

本文选题：TRAIL + 原核表达　；参考：《河南大学》2007年硕士论文

【摘要】： TRAIL(TNF-related apoptosis-inducing ligand)是属于TNF超家族的二型跨膜蛋白,在正常人体的器官和组织中广泛表达。它能够引起包括肿瘤细胞及一些转化细胞的凋亡。TRAIL和TNFα及FasL有非常高的同源性。但是TNFα及FasL分别因为能够引起致命性的炎症反应,导致脓毒性休克症和引起致死性肝衰竭而限制了其在临床上的应用。TRAIL由于能够选择性的诱导肿瘤细胞凋亡而在肿瘤生物治疗上有广泛的应用前景。TRAIL也能和传统的放化疗起到协同增强的效应。但是有报道称不同形式的重组TRAIL会在抗肿瘤效应和细胞毒性上有所不同。所以人们一方面在继续关注TRAIL的安全性,另一方面也在积极的寻找TRAIL的替代品。而抗DR4、DR5单抗由于也能够通过和死亡受体结合而向下传递凋亡信号,最终引起靶细胞的凋亡而成为理想的候选者。自2001年TRA-8(抗DR5单抗)面世以来,就由于其良好的抗肿瘤活性而且对正常组织细胞没有毒性给人们很大的信心。近年来对于抗DR4,DR5的抗体,国内外进行了很多的研究,但是至今还没有上市的药物。本研究就是一方面通过分子生物学的方法实现两种不同形式的人源TRAIL胞外段结构域( 114- 281aa)在大肠杆菌中的可溶性表达,一种是在其氨基末端加上多聚组氨酸标签和S-Tag(TRAIL-His);另一种是不加任何的外源标签蛋白(TRAIL-NH),并初步比较了两种TRAIL蛋白的功能。RT-PCR法从人外周血单个核细胞中扩增TRAIL胞外段基因,克隆入pGEM-T-Easy载体, DNA序列测定鉴定正确性。然后分别将其克隆入原核表达载体pET-30a(His)/pET-28a(NH)。采用E.coli BL21(DE3)表达, Ni亲和柱和离子柱分离纯化目的蛋白。SDS-PAGE和Western blot鉴定原核表达产物。MTT法、PI染色法及瑞氏-姬姆萨染色法观察TRAIL-His,TRAIL-NH蛋白的生物学活性。所表达目的蛋白分子量与预期蛋白分子量相一致,该蛋白可以与抗TRAIL多克隆抗体及抗组氨酸标签抗体反应,并可抑制人淋巴瘤细胞系Jurkat细胞的增殖和诱导Jurkat细胞凋亡。另一方面对本室获得的一株抗DR5单抗mDRA6进行人源化改造以扩大其应用前景。采用分子生物学的方法首先对抗体进行嵌合化改造。首先用RT-PCR法从分泌mDRA6的杂交瘤中钓取可变区基因,克隆入pGEM-T-Easy载体,经测序后,在BLAST上进行比对证实为新的鼠源抗体。然后分别构建嵌合抗体c-mDRA6的轻重链真核表达载体VHexpress、VKexpress,酶切鉴定正确后,脂质体法转染宿主细胞293T,经72小时培养后,检测上清表达量, c-mDRA6表达量为490ng/ml.成功的实现了真核表达。
[Abstract]:TRAIL(TNF-related apoptosis-inducing ligandandis a type of transmembrane protein belonging to TNF superfamily, which is widely expressed in normal human organs and tissues. It can induce apoptosis of tumor cells and some transformed cells. Trail and TNF 伪 and FasL have very high homology. But TNF 伪 and FasL cause fatal inflammatory reactions, respectively. Septic shock and fatal liver failure limit its clinical application. Trail can be widely used in tumor biotherapy because of its ability to selectively induce tumor cell apoptosis. Trail can also be used in cancer biotherapy Radiotherapy and chemotherapy have synergistic effect. But different forms of recombinant TRAIL have been reported to differ in anti-tumor effects and cytotoxicity. So on the one hand people continue to focus on the security of TRAIL, on the other hand, they are actively looking for alternatives to TRAIL. The anti DR4 DR5 McAb is an ideal candidate for apoptosis due to its ability to pass down the apoptosis signal by binding to the death receptor and eventually to induce the apoptosis of the target cells. Since the introduction of TRA-8 (anti DR5 monoclonal antibody) in 2001, people have been confident because of its good antitumor activity and no toxicity to normal tissue cells. In recent years, a lot of research on anti-DR4 DR5 antibody has been carried out at home and abroad, but there is no drug on the market up to now. In this study, two different forms of human TRAIL extracellular domain (114-281aa) were expressed in E. coli by molecular biology. One is to add polyhistidine tag and S-Tagnil Hishe tag to its amino terminal, the other is to add no exogenous tag protein, TRAIL-NH, and to compare the function of two kinds of TRAIL proteins. RT-PCR was used to amplify the extracellular TRAIL gene from human peripheral blood mononuclear cells (PBMC). The pGEM-T-Easy vector was cloned and the DNA sequence was identified correctly. Then they were cloned into the prokaryotic expression vector pET-30aHis / pET-28aHN. The biological activity of TRAIL-His-TRAIL-NH protein was observed by E.coli BL21TDE3 expression, Ni affinity column and ion column separation and purification of the target protein. SDS-PAGE and Western blot were used to identify the prokaryotic expression product. The bioactivity of TRAIL-His-TRAIL-NH protein was observed by Pi staining and Reich's Giemsa staining. The molecular weight of the expressed target protein is consistent with that of the expected protein. The protein can react with anti TRAIL polyclonal antibody and anti histidine label antibody, and can inhibit the proliferation of human lymphoma cell line Jurkat and induce apoptosis of Jurkat cells. On the other hand, a strain of anti DR5 McAb mDRA6 obtained in our laboratory was humanized to expand its application prospect. The chimerism of antibody was first modified by molecular biology. The variable region gene was isolated from the hybridoma secreting mDRA6 by RT-PCR method and cloned into the pGEM-T-Easy vector. After sequencing, it was confirmed to be a new murine antibody by comparison on BLAST. Then, the eukaryotic expression vector of chimeric antibody c-mDRA6 was constructed respectively. After restriction endonuclease digestion, 293T was transfected into host cells by liposome method. After 72 hours of culture, the expression of supernatant was detected, and the expression of c-mDRA6 was 490ng / ml. The eukaryotic expression was successfully realized.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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