重组逆转录病毒载体pLXPXSN-TCRα12-2-IRES-Vβ7.1的构建及其杀伤肝癌细胞作用的研究

Construction of Recombinant Retrovirus Vector pLXPXSNTCRα12-2-IRESVβ7.1 and Study of the Cytotoxicity to Hepatoma Cell

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 dicistronic retrovirus expression vector to coxpress both human TCRα12-2 and TCRVβ7.1genes and transfect PBMC on virus. Methods： The TCRα12-2 and TCRVβ7.1 were amplified by PCR from plamid that we constructed before, which was cloned into vector pLXPXSN. The recombinant plasmid pLXPXSN- TCRα 12-2-IRES- Vβ7.1 was verified by restriction enzyme digestion. Then the positive recombinant plasmid was transferred into PA317 cells using Lipofectamine 2000, and the titer was measured. Then the protein TCR was detected by flow cytometry. The effect of retrovirus on BEL-7402 cells and HepG2 cell was measured by flow cytometry and MTT assay. Results： The dicistronic expression vector pLXPXSN- TCRα12- 2-IRES-Vβ7.1 was successfully constructed and expressed. The killing rate of the PBMC infected by pLXPXSN-TCRα 12-2-IRES- Vβ7.1 was obviously higher than the other group. Conclusion： The TCRα 12-2 and TCRVβ 7.1 were integrated into the host genome and expressed.The killing effect of the PBMC infected by pLXPXSN-TCRα 12-2-IRES-Vβ 7.1 for hepatoma cells is improved.