hTERT-逆转录病毒载体构建及其在脐血间质干细胞中的表达

Construction of recombinant retrovirus vector carrying hTERT and expression MSCs in human cord blood

目的:构建hTERT-逆转录病毒载体,向脐血间质干细胞(UCBMSCs)中导入hTERT基因,观察该基因的表达。方法:PCR法扩增出hTERT全部片断,定向克隆入pLNCX2载体。将病毒质粒导入包装细胞内,筛选阳性产毒细胞,测定病毒滴度。取病毒上清感染脐血MSCs,筛选转基因细胞,检测转染前后hTERT在mRNA水平的表达。结果:用BglⅡ和NotⅠ双酶切构建质粒,证明PLNCX2-hTERT构建成功,转基因细胞的hTERT基因在mRNA水平得到表达。结论:hTERT-逆转录病毒载体构建成功,在逆转录病毒介导下,外源性hTERT基因转入脐血MSCs后得到表达。

Objective： To construct a recombinant retrovirus vector carrying hTERT gene, and to observe the expression of the genes after UCBMSCs were trasduced into hTERT genes. Methods： The whole cDNA was generated by PCR amplifications from the plasm;d pEGFP - hTERT - C1, then the hTERT segments were subcloned into pLNCX2. These retroviral particles were transduced into target cells. We selected the stably positive virus -producing cells, etermined the titration degree of virus. From virus infected umbilicus blood MSC we selected the transgenetic cells and detected the expression of hTERT in mRNA level by RT - PCR. Results： The constructed plasmids were digested with restriction endonucleases（ Bgl Ⅱ and Not I ） which indicated that PLNCX2 - hTERT were successfully established. The hTERT genes of transgenetic cells were expressed at mRNA level. Conclusion： hTERT - reverse transcription virus carriers were successfully established. Induced by reverse transcription virus, exterior hTERT genes were expressed in UCBMSCS.