负载hTERT基因片段的慢病毒的包装及表达

Package and expression of lentivirus encoding hTERT gene

目的构建编码人端粒酶逆转录酶(hTERT)的慢病毒载体,并测定其滴度,观察hTERT基因在体外的表达情况。方法采用RT-PCR获得hTERT基因片段,插入转移载体L166,用CaCl2法L166-hTERT、L205、L311共转染293T细胞包装慢病毒,测定病毒滴度,免疫细胞化学法检测慢病毒感染的293T细胞中hTERT的表达。结果测序结果显示成功构建了重组质粒L166-hTERT,测定未经浓缩的病毒滴度为5.25×106IU/mL,免疫细胞化学法检测到慢病毒感染的293T细胞中hTERT呈阳性表达。结论成功构建了负载hTERT基因片段的慢病毒,hTERT能在感染的293T细胞中高效表达,表达持续2个月以上。

Objective To construct a lentiviral vector encoding human telomerase reverse transcriptase（hTERT）genes. Methods A fragment of the hTERT gene was amplified by RT-PCR and was inserted into the plasmid L166. The three plasmids L166- hTERT, I205 and I311 co-transfected the virus packaging cell line 293T using the CaC12 method. Then the cell supernatant was harvested and virus titration was determined. Also the expression of hTERT in 293T cells infected by the lentivirus was determined by using the immunocytochemistry method. Results DNA sequencing demonstrated that the recombinant plasmid L166-hTERT was successfully constructed. The titration of the un-concentrated lentivirus was 5.25 ×10^6 IU/mL. hTERT polypeptid immunocytochemistry staining proved positive for the hTERT gene in infected 293T cells. Conclusion Lentivirus encoding hTERT was successfully packaged and hTERT could express in 293T cells for more than 2 months.