细菌内同源重组构建含人内皮抑素基因腺病毒及在胃癌细胞中的体外转染实验

Constructing Recombinant Adenovirus Expressing Human Endostatin Gene by Homologous Recombination in Bacteria

目的 :采用细菌内同源重组法构建含人内皮抑素基因的重组腺病毒Ad -CMV -hEn。方法 :将hEn基因自 pCA13-hEn质粒中酶切下来 ,亚克隆至腺病毒穿梭质粒中 ,形成转移质粒 pAdtrack -CMV -hEn ,并用PmeⅠ线性化后与腺病毒基因组质粒 pAdeasy共转化大肠杆菌BJ5 183,抽提重组体腺病毒基因组质粒DNA ,PacⅠ酶切后在 2 93细胞中包装成腺病毒颗粒。采用PCR方法对重组体腺病毒进行鉴定 ,同时测定了病毒滴度。并采用RT -PCR检测感染腺病毒的胃癌细胞内有无hEnmRNA的转录。结果 :由 pAdtrack -CMV -hEn和pAdeasy共转化大肠杆菌BJ5 183后 ,可得到阳性重组体细菌克隆 ,包装完成的腺病毒经PCR检测表明已含有hEn基因。纯化所得腺病毒滴度约为 2 .4× 10 11pfu /ml。RT -PCR证实在感染重组体腺病毒Ad .CMV -hEN的细胞中有相应mRNA的转录。结论 :细菌内同源重组法是一种高效、简便、快捷的重组体腺病毒载体制备方法。所构建的Ad -CMV -hEn在体外能有效转录相应的mRNA。

Objective: To produce recombinant adenovirus by using a high efficient method of homologous recombination in bacteria with human endostatin as target gene. Methods: hEn gene was from plasmid of pCA13-hEn and cloned into shuttle plasmid and formed transfer plasmid of pAdtrack-cmv-hEn. After linealized with PmeⅠ and co-transformed into BJ5183 bacterial cells with adenovirus genomic DNA plasmid of pAdeasy. The identified recombinant adenovirus plasmid DNA was digested with PacⅠand transfected to 293 cells for package of recombinant adenovirus particles. The PCR technique was used to detect target gene. The titer of the recombinant Ad was measured. RT-PCR proved target gene expression in infected tumor cells. Results: Positive recombinant bacterial clones have been found after co-transformation of BJ5183 bacterial cells. Recombinant adenovirus was obtained from 293 cells transfected with Pac I-digested Ad genomic plasmid DNA. PCR test indicated that the recombinant Ad contained the hEn gene. The titre of purified Ad was 2.4×10 11pfu /ml. Meanwhile, mRNA product of hEn was detected in the infected tumor cells by RT-PCR. Conclusion: The method of homologous recombination in bacteria is a convenient and high efficient method to produce recombinant adenovirus, and the recombinant adenoviruses can effectively mediate target gene expression in infected SGC-7901 cells.