人端粒酶逆转录酶永生化MRC-5细胞方法的建立

Preparation of human telomerase reverse transcriptase-immortalized MRC-5 cells

目的 通过携带人端粒酶逆转录酶（hTERT）的慢病毒颗粒转染法,制备可用于人巨细胞病毒（HCMV）培养的永生化MRC-5细胞.方法 以携带hTERT基因的质粒pLXSN-hTERT为分子基础,通过PCR构建慢病毒载体质粒pLVX-EFl a-hTERT.pLVX-EF1 a-hTERT质粒与Lenti-X HTXPackaging Mix2混合后转染293T细胞,并经Real-time PCR定量所获得的慢病毒颗粒,在慢病毒原液感染MRC-5细胞后连续传代获得永生化的MRC-5T细胞,并观察HCMV在MRC-5T细胞中的感染特性.结果 获得了表达hTERT基因的慢病毒颗粒LVX-EF1a-hTERT,制备了可用于HCMV培养的永生化MRC-5细胞.结论 建立了人端粒酶逆转录酶永生化MRC-5细胞的方法,为永生化原代细胞等其他种类细胞提供了技术保障和物质基础.

Objective To prepare immortalized MRC-5 cells by transduction of human telomerase reverse transcriptase gene （hTERT）,which can be used for the routine propagation of human cytomegalovirus （HCMV）.Methods The recombinant plasmid pLVX-EFla-hTERT was constructed by cloning hTERT gene into the vector pLVX-EFla-Tet3G,and hTERT gene was amplified from another plasmid pLVX-EFla-Tet3G.Lentvirus particles LVX-EF1a-hTERT were prepared by co-transfected 293T cells with pLVX-EF1 a-hTERT and Lenti-X HTX Packaging Mix.After that,Lentvirus particles were used to infect MRC-5 cells,then both the propagation and expression of hTERT in the passaged MRC-5T were examined.Results We obtained Lentvirus particles LVX-EF1a-hTERT and immortalized MRC-5 cells.Conclusion Method of human telomerase reverse transcriptase-immortalized MRC-5 cells will contribute to the construction of immortalized primary cells.