【摘要】 目的:构建G-四链体抗体BG4基因的真核表达载体p EGFP-N1-BG4,探讨转染该真核表达载体致BG4蛋白过表达对人胃癌细胞株AGS凋亡和端粒酶逆转录酶表达的影响。方法:以BG4基因原核表达载体p SANG10-BG4为模板,PCR扩增出含有BG4基因的目的片段,经TA克隆与p MD18-T载体连接,测序正确后,经Sac I和Pst I双酶切,与真核表达载体p EGFP-N1连接后转化感受态大肠杆菌DH5α,并进行酶切分析和序列测定,Western blot鉴定BG4蛋白表达;流式细胞术检测细胞周期;DAPI法及HE染色检测各组细胞凋亡;q PCR和Western blot检测空白对照组、p EGFP-N1组和p EGFP-N1-BG4组端粒酶逆转录酶mRNA和蛋白表达的变化。结果:酶切分析显示,目的基因条带与预期结果相符,DNA测序表明克隆的BG4基因序列正确,Western blot结果表明转染p EGFP-N1-BG4质粒可以成功表达BG4蛋白。转染p EGFP-N1-BG4质粒可抑制细胞进入S期。DAPI法及HE染色显示,p EGFP-N1-BG4组出现明显的新月形核和核固缩的细胞凋亡现象。q PCR和Western blot结果表明,p EGFPN1-BG4组端粒酶逆转录酶表达受到抑制。结论:用p EGFP-N1-BG4真核表达载体转染胃癌细胞株AGS可以抑制该细胞端粒酶逆转录酶表达,诱导细胞凋亡。更多还原

【Abstract】 AIM: To construct the eukaryotic expression vector p EGFP-N1-BG4 for G-quadruplex antibody BG4,and to evaluate its effect on the apoptosis and expression of telomerase reverse transcriptase( TERT) in human gastric cancer cell line AGS. METHODS: The prokaryotic expression vector p SANG10-BG4 for BG4 gene previously constructed in our group was used as a template. The target fragment containing BG4 gene was amplified by PCR and then ligated with p MD18-T vector via TA cloning. After sequencing and Sac I and Pst I double digestion,eukaryotic expression vector p EGFP-N1 was transformed into competent E. coli DH5α,and then double enzyme digestion analysis and sequencing were performed. Western blot was used to identify BG4 protein expression. q PCR and Western blot were used to detect the expression of TERT in control group,p EGFP-N1 group and p EGFP-N1-BG4 group. DAPI method was used to detect apoptosis in each group. RESULTS: Enzyme digestion analysis showed that the band of the target gene was consistent with the expected result. DNA sequencing demonstrated that the sequence of the cloned BG4 gene was correct. The expression of BG4 protein was conformed by Western blot,indicating that the transfection of p EGFP-N1-BG4 into the AGS cells was successful. The results of q PCR and Western blot showed that the expression of TERT in p EGFP-N1-BG4 group was inhibited.DAPI and HE staining showed that there was obvious apoptosis phenomenon in p EGFP-N1-BG4 group as indicated by crescentic nucleus and nuclear pyknosis. CONCLUSION: The eukaryotic expression vector p EGFP-N1-BG4 is constructed successfully. Transfection of the vector into AGS cell line can inhibit the TERT expression and induce cell apoptosis.更多还原