hTERT重组第三代慢病毒载体的构建及其病毒包装鉴定

Construction of Recombinant Telomerase Reverse Transcriptase Gene Lentiviral Expression Vector and Virus Packaging

目的:构建携带人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)基因的慢病毒表达载体及探索高滴度第三代慢病毒包装体系的建立,并观察hTERT基因在细胞中的表达调控。方法:将hTERT基因片段插入慢病毒载体pCDH-CMV-MCS-EF1-copGFP中,构建慢病毒表达质粒pCDH-hTERT。通过酶切、DNA测序验证hTERT片段的准确性后,将pCDH-hTERT、pCDH-PACK-GAG、pCDH-PACK-REV和VSV-G共转染包装细胞293T,浓缩上清并测定病毒滴度获得重组慢病毒,并通过PCR及293T中hTERT蛋白的表达鉴定重组慢病毒。将重组慢病毒感染靶细胞,通过检测标记蛋白-绿色荧光蛋白(green fluorescent protein,GFP)、hTERT蛋白表达和端粒酶活性进一步验证pCDH-hTERT在细胞中表达。结果:pCDH-hTERT携带正确hTERT基因,将其与包装质粒共转染293T细胞能产生重组病毒;病毒基因组PCR证实hTERT基因插入,感染293T后可检测到hTERT蛋白的表达;目的基因hTERT能被重组慢病毒高效地转导入靶细胞并稳定表达,荧光显微镜下可直接观察GFP;RT-PCR、Western blot及TRAP-PCR-ELISA法检测感染后的细胞,发现hTERTmRNA、hTERT蛋白的表达及端粒酶活性明显增强。结论:成功构建了第三代慢病毒表达载体pCDH-hTERT,并获得高效的重组慢病毒,能将外源hTERT基因导入靶细胞重建端粒酶活性,为构建永生化细胞系奠定基础。

Objective: The aim is to construct lentiviral vector containing GFP reporter gene driven by human telomerase reverse transcriptase(hTERT)gene and to establish high-titer lentiviral packaging system,then observe the expression of hTERT gene.Methods: Digested the plasmid PCI-neo-hTERT with double enzyme digestion and subcloned hTERT into the lentiviral vector,pCDH-CMV-MCS-EF1-copGFP,to generate the lentiviral expression vector,pCDH-hTERT.The accuracy of hTERT fragment was confirmed by double enzyme digestion and DNA sequencing analysis.Recombinant lentiviruses were produced by 293T cells following the co-transfection of pCDH-hTERT,with the packaging plasmids pCDH-PACK-GAG、pCDH-PACK-REV and VSV-G which are the 3rd generation of lentiviral vector systems containing green fluorescent protein(GFP)gene.Virus supernatant was collected and concentrated,then the titer of virus was tested.The resulting recombinant lentiviruses which carrying hTERT were then used to infect target cell lines.GFP,hTERT mRNA and telomerase expression in 293T and hUVEC were detected by fluorescent microscope,RT-PCR,Western blotting and TRAP-PCR-ELISA.Result: Plasmid pCDH-hTERT carried the correct hTERT gene.The recombinant lentiviruses pCDH-hTERT which carried hTERT could be produced by co-transfection of pCDH-hTERT and packaging plasmids to 293T;The recombinant lentiviruses which carried hTERT could infect and deliver hTERT gene to 293T and hUVEC,and hTERT mRNA and protein expression were remarkably increased in infected cells.Conclusion: The study successfully constructed recombinant plasmid pCDH-hTERT,the recombinant lentiviruses can deliver target gene hTERT and have high infection efficiency.Extrinsic hTERT gene can reconstruct telomerase and settle a basis for establishing immortalized fibroblast of cells.