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目的:构建一个表达胃癌相关基因GCRG213小干扰RNA的重组腺相关病毒载体,制备能靶向性抑制GCRG213表达的重组腺相关病毒.方法:将构建好并经验证的包含胃癌相关基因GCRG213特异性小干扰RNA片段GCRG213-RNAi-2的IMG800-GCRG213i-2质粒用Bam H I+Bg/Ⅱ双酶切,回收410 bp左右目的片段,将pSNAV2.0质粒用BamH I单酶切后同目的片段连接,经酶切和测序鉴定后,用新建质粒脂质体转染BHK-21细胞,G418筛选后大规模培养,再用辅助病毒HSV1-rc/△UL2感染,获取病毒.结果:成功构建包含U6启动子和胃癌相关基因GCRG213特异性RNA干扰片段的重组腺相关病毒载体,并制备出滴度为5×10~(12)vector genome/L高滴度腺相关病毒.结论:载体的构建为进一步研究GCRG213的体内功能打下了基础,也将为更多的基因干预和基因的功能研究提供有效的工具.
OBJECTIVE: To construct a recombinant adeno-associated virus vector expressing small interfering RNA of gastric cancer related gene GCRG213 and prepare a recombinant adeno-associated virus that can inhibit the expression of GCRG213.Methods: The constructed and validated recombinant adeno-associated virus (GCRG213) Interfering RNA fragment GCRG213-RNAi-2 IMG800-GCRG213i-2 plasmid with Bam H I + Bg / Ⅱ double digestion, recovery of about 410 bp fragment of interest, the pSNAV2.0 plasmid BamH I single digestion after the same fragment After identification by restriction enzyme and sequencing, BHK-21 cells were transfected with the constructed plasmid liposomes, and then screened with G418 for large-scale cultivation and then infected with virus HSV1-rc / △ UL2 to obtain the virus.Results: Promoter and gastric cancer associated gene GCRG213 specific RNA interference fragment of recombinant adeno-associated virus vector and prepared with a titer of 5 × 10-12 vector genome / L high titer adeno-associated virus.Conclusion: The vector was constructed further The study of GCRG213 in vivo function laid the foundation, but also for more gene intervention and gene function research provides an effective tool.