HIF-1α基因慢病毒载体的构建和鉴定

Construction and Identification of HIF-1α Gene Recombinant Lentivirus Vector

[目的]构建带有大鼠HIF-1α基因的慢病毒表达载体,并进行病毒颗粒的包装与对293T的转染。[方法]从pEGFP-N1-HIF-1α载体中扩增出HIF-1α基因片段,将目的基因与pLenti6.3-MCS-RES2-EGFP载体连接,获得慢病毒表达载体pLenti6.3-HIF-1α-IRES2-EGFP,并用磷酸钙转染法将其与慢病毒包装系统共同转染到293T细胞内进行病毒包装,48h后收集上清液并过滤,按不同感染复数感染293T细胞,通过观察报告基因绿色荧光蛋白的表达来测定病毒滴度和感染效率。[结果]构建了共表达HIF-1α基因和EGFP基因的重组慢病毒载体pLenti6.3-HIF-1α-IRES2-EGFP,经PCR及测序结果证实,并对其成功包装出慢病毒,病毒滴度为4×106TU/ml。[结论]成功构建出HIF-1α基因重组慢病毒载体pLenti6.3-HIF-1α-IRES2-EGFP并能有效的包装出慢病毒。

[Objective] To construct lentiviral vector which encodes full length rat HIF-1α gene and package the virus particles.[Methods] The HIF-1α gene was amplified from pEGFP-N1-HIF-1α vector and cloned into a pLenti6.3-MCS-RES2-EGFP lentiviral vector to obtain the lentiviral expressing vector pLenti6.3-HIF-1α-IRES2-EGFP.293T cells were used as packaging cells.The target plasmid pLenti6.3-HIF-1α-IRES2-EGFP,together with packaging plasmids were co-transfected into 293T cells using calcium phosphate transfection method.After 48 h,cell culture supernatant was collected and filtrated.Then 293T cells were exposed to lentivirus with different multiplicity of infection.Transfection efficiency was estimated by evaluation of the green fluorescent protein expression via fluorescence microscopy.[Results] The recombinant lentiviral vector pLenti6.3-HIF-1α-IRES2-EGFP which co-expressed the HIF-1α and EGFP gene was constructed and verified by PCR and sequencing.The titer of the packaged lentiviral virus was 4×106TU/ml.[Conclusion] The recombinant lentiviral vector pLenti6.3 HIF-1α-IRES2-EGFP was constructed successfully and could be effectively used to package the lentiviral.