CAJ | 학술논문

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.
목적 통과휴대인단립매역전록매(hTERT)적만병독과립전염법,제비가용우인거세포병독(HCMV)배양적영생화MRC-5세포.방법 이휴대hTERT기인적질립pLXSN-hTERT위분자기출,통과PCR구건만병독재체질립pLVX-EFl a-hTERT.pLVX-EF1 a-hTERT질립여Lenti-X HTXPackaging Mix2혼합후전염293T세포,병경Real-time PCR정량소획득적만병독과립,재만병독원액감염MRC-5세포후련속전대획득영생화적MRC-5T세포,병관찰HCMV재MRC-5T세포중적감염특성.결과 획득료표체hTERT기인적만병독과립LVX-EF1a-hTERT,제비료가용우HCMV배양적영생화MRC-5세포.결론 건립료인단립매역전록매영생화MRC-5세포적방법,위영생화원대세포등기타충류세포제공료기술보장화물질기출.
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.