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在肿瘤免疫治疗和基因治疗的研究中,尽管CTL的诱导至关重要,目前尚没有办法能够直接研究肿瘤特异性CTL的产生和作用.Tetramer是一种最新的能直接识别CTL上TcR的工具.我们通过设计合适的引物,在H-2K~b胞外段引人生物素特异结合多肽BSP,将H-2K~b-BSP克隆到pET21d表达载体.将表达H-2K~b和β_2微球蛋白的质粒转化E.coliBL21(DE3),分别制备H-2K~b和β_2微球蛋白的包含体.将E.G7细胞的特异性抗原多肽OVA_(257～264)、处于还原状态H-ZK~b的包含体、β_2微球蛋白的包含体三者一起通过稀释复性,复性后的多肽/H-2K~b/β_2微球蛋白聚合体经浓缩后过Sephacryl S200分子筛层析纯化.纯化后的单分子多肽/H-2K~b/β_2微球蛋白复合体在Bir A酶的作用下与生物素连接.
In the study of tumor immunotherapy and gene therapy, although the induction of CTL is critical, there is currently no way to directly study the generation and role of tumor-specific CTL. Tetramer is a new tool that can directly identify TcR on CTL. We designed a suitable primer to introduce the biotin-specific binding polypeptide BSP in the extracellular domain of H-2K~b, and cloned H-2K~b-BSP into the pET21d expression vector. The H-2K~b and β_2 microspheres will be expressed. The plasmid of the protein was transformed into E. coli BL21 (DE3) to prepare H-2K~b and β2 microglobulin inclusion bodies respectively. The specific antigen polypeptide OVA_(257~264) of E.G7 cells was in the reducing state H-ZK. The inclusion bodies of ~b and the inclusion bodies of β 2 -microglobulin were diluted and renatured together. The renatured polypeptide/H-2K~b/β 2 microglobulin polymer was concentrated and purified by Sephacryl S200 molecular sieve chromatography. The purified single-molecule polypeptide/H-2K~b/beta-2 microglobulin complex was linked to biotin by the action of Bir A enzyme.