胃癌相关基因GCRG 213正反义表达重组腺相关病毒载体的构建

Construction of Recombinant Adeno-associated Virus Vector Expressing Sense or Anti-sense Gene GCRG 213 Related to Gastric Cancer

目的构建胃癌相关基因OCRG 213正、反义表达重组腺相关病毒载体,制备相应的重组腺相关病毒。方法通过引入限制性内切酶识别位点从pGEM-T质粒上扩增出胃癌相关基因GCRG 213的DNA片段,按正、反向克隆入pCMV-MCS载体,测序正确后用NotI酶切,克隆入pAAV-MCS载体,单、双酶切鉴定后,同pAAV-Helper、pAAV- RC质粒用磷酸钙转染法共转染AAV-293细胞制备病毒。结果成功构建包含胃癌相关基因GCRG213正、反义克隆的重组腺相关病毒载体,并制备出滴度为4×1010v．g．／ml腺相关病毒。结论载体的构建和病毒制备为进一步研究GCRG 213的体内功能打下了基础。

Objective To construct the recombinant adeno-associated virus（AAV） expressing vector sense or antisense gene C-CRG 213 related to gastric cancer. Methods The sense and anti-sense fragments of gene GCRG 213 related to gastric cancer, which was introduced into the sites of restrictive endonuclease enzyme BamHI plus PstⅠ, and BamHI plus ClaI, were obtained by PCR and cloned into AAV vectors respectively. After identification by PCR, restrictive endonuclease enzyme digestion and sequencing, the recombinant plasmid was co-transfected into AAV 293 cells with pAAV-Helper and pAAV-RC plasmids by calcium phosphate precipitation. Results The recombinant AAV vectors expressing sense or anti-sense gene C-CRG 213 related to gastric cancer were constructed successfully and 4 ×10^10μg/ml recombinant AAV was obtained. Conclusion Recombinant AAV vectors can be used for further research into C-CRG 213 function in vivo.