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目的 构建融合基因OVA2 57 2 64 β2m的表达载体 ,并以其表达产物作为免疫原 ,在体内诱导特异性CTL。方法 采用PCR技术 ,从人肝癌细胞株SMMC 772 1中克隆人β2m基因 ,拼接上OVA(2 5 7～ 2 6 4)序列及linker后 ,克隆入pET 42b(+)高效表达载体进行诱导表达。对表达产物进行纯化与复性后 ,用其作为免疫原诱导特异性CTL ,并构建H 2Kb OVA四聚体检测特异性CTL。结果 成功地获得融合蛋白OVA2 57 2 64 β 2m及H 2Kb OVA四聚体 ,应用H 2Kb OVA四聚体对特异性CTL检测结果与经典的细胞毒试验一致。结论 OVA2 57 2 64 β 2m的表达产物 ,可有效的在体内诱导特异性CTL应答。在体外构建的MHCI类分子四聚体 ,为特异性T细胞的检测提供了有利的工具
Objective To construct an expression vector of fusion gene OVA2 57 2 64 β2m and use its expressed product as an immunogen to induce specific CTL in vivo. Methods The human β2m gene was cloned from human hepatocellular carcinoma cell line SMMC 772 1 by PCR and cloned into pET 42b (+) vector after splicing with OVA (257 ~ 264) sequence. After purification and refolding of the expressed product, it was used as an immunogen to induce specific CTLs, and a H 2Kb OVA tetramer was constructed to detect specific CTLs. Results The fusion proteins OVA2 57 2 64 β 2m and H 2Kb OVA tetramers were successfully obtained. The results of the specific CTL assay using H 2Kb OVA tetramers were consistent with the classical cytotoxicity assays. Conclusion The expression product of OVA2 57 2 64 β 2m is effective in inducing specific CTL responses in vivo. The in vitro MHC class I tetramers provide an advantageous tool for the detection of specific T cells