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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 positive and negative antisense recombinant adeno-associated virus vectors of gastric cancer related gene OCRG 213 and prepare the corresponding recombinant adeno-associated virus. Methods DNA fragment of gastric cancer related gene GCRG 213 was amplified from plasmid pGEM-T by introducing restriction endonuclease recognition sites. The DNA fragment was cloned into pCMV-MCS vector in both forward and reverse directions. After sequencing correctly, it was digested with NotI and cloned The recombinant plasmid pAAV-MCS was transfected into pAAV-MCS vector. After single and double enzyme digestion, the recombinant plasmid pAAV-Helper and pAAV-RC were co-transfected into AAV-293 cells by calcium phosphate transfection. Results The recombinant adeno-associated virus vector containing positive and antisense clone of gastric cancer related gene GCRG213 was successfully constructed and the titer was 4 × 1010v. g. / Ml adeno-associated virus. Conclusion The construction of vector and preparation of virus laid the foundation for further study on the function of GCRG 213 in vivo.