人HIF1α基因重组逆转录病毒载体构建及鉴定

Construction and identification of human hypoxia-inducible factor-1α gene recombinant retrovirus

目的 构建人缺氧诱导因子 1α(HIF 1α)基因重组逆转录病毒载体 ,并观察其对NIH3T3细胞感染效率。方法 采用基因工程技术 ,经过 2次亚克隆将HIF 1α基因片段克隆至含IRES EGFP逆转录病毒载体上 ,鉴定后用脂质体法转染PT67细胞进行包装、扩增 ,最后用重组逆转录病毒感染NIH3T3细胞。其中采用PCR方法对重组载体进行鉴定 ,利用绿色荧光蛋白作为报告基因 ,对病毒滴度和感染效率进行检测。结果 酶切鉴定及PCR结果与HIF 1α基因重组逆转录病毒载体的预期结果一致 ,病毒滴度达 ( 1 4× 10 6pfu/ml) ,并对NIH3T3细胞有强感染能力。结论 应用基因工程技术 ,成功构建了含人HIF 1α基因重组逆转录病毒载体 。

Objective To construct the recombinant retrovirus of human hypoxia inducible factor 1α(HIF 1α) gene and to observe its ability to infect NIH3T3 fibroblasts. Methods The full length human HIF 1α cDNA was cloned into the retroviral vector containing internal ribosomal entry site (IRES) and enhanced green fluorescent protein (EGFP) by the method of gene engineering. Then the retroviral vector with HIF 1α was transfected into PT67 cells using the lipofectine DOTAP and NIH3T3 cells was infected by the recombinant retrovirus. The target gene was detected by polymerase chain reaction (PCR). The titer and its infection rate were determined using the EGFP expression with a fluorescent microscope. Results Restriction endonuclease and PCR analyses confirmed that the human HIF 1α cDNA was successfully inserted into the retroviral vector. The titer of recombinant retrovirus with HIF 1α gene was 1 4×10 6 pfu/ml and the retrovirus had a strong effect on NIH3T3 cells. Conclusion The recombinant retrovirus containing HIF 1α gene has been successfully constructed by the method of gene engineering, which lays a foundation for the application in therapeutic angiogenesis.