pcDNA3.1-GCNF真核表达载体构建及其对HeLa细胞凋亡的影响

Construction of pcDNA3.1-GCNF Eukaryotic Expression Vector and Effects of Apoptosis in HeLa Cells

构建人pcDNA3.1-GCNF真核表达载体,并瞬时转染宫颈癌HeLa细胞,观察GCNF基因对HeLa细胞凋亡的影响。采用PCR的方法从pMD18-T-GCNF载体中扩增全长GCNF cDNA,将扩增产物克隆至真核表达载体pcDNA3.1(-),测序鉴定后,重组质粒pcDNA3.1-GCNF转染HeLa细胞,RT-PCR以及间接免疫荧光鉴定转染结果,并用Annexin V-PI双染流式细胞术分析细胞凋亡情况。结果显示,克隆的重组真核表达载体经序列测定分析后,证实pcDNA3.1-GCNF重组真核表达质粒构建成功,并获得瞬时转染pcDNA3.1-GCNF的HeLa细胞。流式细胞术检测:转染重组质粒组的细胞凋亡率(6.72%)低于转染空质粒组(9.78%)以及未转染组(9.34%),P<0.00313,转染重组质粒后细胞凋亡率降低。由此可知,pcDNA3.1-GCNF重组质粒瞬时转染子宫颈癌HeLa细胞后,GCNF基因可以抑制HeLa细胞凋亡。

To construct the pcDNA3.1-GCNF eukaryotic expression vector and transient transfection into cervical cancer HeLa cells and observe the influence of GCNF genes on HeLa cells apoptosis,PCR method was used to amplify GCNFcDNA from the pMD18-T-GCNF vector,the amplification product was cloned into the pcDNA3.1（-） vector and sequencing,the recombinant plasmid pcDNA3.1-GCNF transfected into cervical cancer HeLa cells,RT-PCR and indirect immunofluorescence identified the result of transfected.Analysis apoptosis of HeLa cells by Annexin V-PI double staining flow cytometry.The pCDNA3.1-GCNF recombinant plasmid was successfully constructed and confirmed by DNA sequence.Then the pcDNA3.1-GCNF recombinant plasmid was successfully transfected into cervical carcinoma HeLa cells.Recombinant plasmid transfected group（6.72%） of cell apoptosis lower than empty vector transfected group（9.78%）and control group（9.34%）,（P0.00313）.The pcDNA3.1-GCNF recombinant plasmid was transfected into HeLa cells.GCNF gene could inhibit apoptosis of HeLa cells.