人低氧诱导因子2α野生型慢病毒载体的构建及鉴定

The Construction and Identification of the Hypoxia-inducible Factor-2α Wild-type Lentiviral Expression Vector

目的:构建乏氧诱导因子-2α野生型慢病毒表达载体及鉴定。方法:用PCR扩增人HIF-2α基因片段与经酶切后plvx-EGFR-3-FLAG-Puro表达载体片段同源重组后转化E.coli感受态细胞。用菌落PCR鉴定转化子,阳性克隆鉴定,经病毒包装,感染人胃癌MGC-803细胞,采用QPCR、Western blotting检测HIF-2α基因的表达。结果:构建HIF-2α基因野生型慢病毒表达载体QPCR、Western blotting检测HIF-2α基因的表达均上调,高于对照组,P<0.05。结论:成功构建HIF-2α基因野生型慢病毒表达载体,为进一步研究HIF-2α在肿瘤发生、发展、侵袭、转移、耐药等的生物学功能提供工具模型。

Objective:The paper is to construct and identify the hypoxia-inducible factor-2α wild-type lentiviral expression vector.Method:The human HIF-2α gene fragment amplified by the PCR was homologously recombined with the plvx-EGFR-3-FLAG-Puro expression vector fragment processed with enzyme digestion to transform E. coli competent cells. The colony PCR was used to identify the transformant and the positive clones were identified. After virus packaging and infecting with the human gastric cancer MGC-803 cells, the expression of HIF-2α gene was detected with the QPCR and the Western Blotting.Results:The expressions of the HIF-2α gene which were detected by the QPCR and the Western Blotting in the constructed HIF-2α gene wild-type lentiviral expression vector were both up regulated, which was higher than the control group(P<0.05).Conclusions:The HIF-2α gene wild-type lentiviral expression vector is successfully constructed to provide a tool model for further exploring the biological functions of the HIF-2α in tumorigenesis, progression, invasion, transfer and drug resistance.