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目的:构建双表达逆转录病毒载体pLXPXSN-TCRα12-2-IRES-Vβ7.1,包装成病毒颗粒后有效地感染PBMC。方法:以实验室保存含TCRVβ7.1基因和TCRα12-2基因的质粒为模板,分别扩增得到两个基因,亚克隆入载体pLXPXSN,得到重组质粒pLXPXSN-TCRα12-2-IRES-Vβ7.1。重组体质粒经酶切鉴定后,将鉴定好的阳性重组质粒用脂质体介导转染PA317细胞,包装成完整的病毒后测定滴度,感染PBMC,用流式细胞仪和提取基因组DNA检测目的蛋白的表达。最后病毒感染PBMC,用流式细胞仪检测目的蛋白的表达。然后用流式细胞术细胞凋亡率,MTT比色法检测pLXPXSN-TCRα12-2-IRES-Vβ7.1感染的PBMC对肝癌细胞BEL-7402和HEPG2的杀伤作用。结果:从重组病毒基因组中扩增出目的基因TCRα12-2和TCRVβ7.1,流式细胞仪检测表明目的基因可以在PBMC中有效的表达。pLXPXSN-TCRα12-2-IRES-Vβ7.1感染PBMC组对肿瘤细胞的杀伤率明显高于PBMC组和空载体感染组。结论:TCRα12-2和TCRVβ7.1能够整合进宿主PBMC的基因组中,并能得到有效地表达。pLXPXSN-TCRα12-2-IRES-Vβ7.1感染PBMC后可提高其对肝癌细胞的杀伤活性。
OBJECTIVE: To construct a double-expression retroviral vector pLXPXSN-TCRα12-2-IRES-Vβ7.1, which effectively infects PBMC after being packaged into virosomes. Methods: Two plasmids containing TCRVβ7.1 gene and TCRα12-2 gene were used as templates in the laboratory, and subcloned into vector pLXPXSN to obtain the recombinant plasmid pLXPXSN-TCRα12-2-IRES-Vβ7.1. Recombinant plasmids were identified by restriction enzyme digestion, the positive recombinant plasmids were identified and transfected into PA317 cells with liposomes, and the titers were determined by packaging into a complete virus. PBMCs were infected and detected by flow cytometry and genomic DNA extraction Expression of the target protein. Finally, the virus infected PBMCs and the expression of the target protein was detected by flow cytometry. Then the cytotoxicity of pLXPXSN-TCRα12-2-IRES-Vβ7.1 infected PBMC on hepatocellular carcinoma cells BEL-7402 and HEPG2 was detected by flow cytometry and MTT assay. Results: The target genes TCRα12-2 and TCRVβ7.1 were amplified from the recombinant virus genome. The results of flow cytometry showed that the target gene could be efficiently expressed in PBMCs. The killing rate of tumor cells by pLXPXSN-TCRα12-2-IRES-Vβ7.1 infected PBMC group was significantly higher than that of PBMC group and empty vector. Conclusion: TCRα12-2 and TCRVβ7.1 can be integrated into the genome of host PBMCs and can be efficiently expressed. Infection of PBMC with pLXPXSN-TCRα12-2-IRES-Vβ7.1 enhanced its cytotoxic activity against hepatoma cells.