细菌内同源重组法构建靶向Survivin的腺病毒载体的研究

Construction of Recombinant Adenoviral Vector Carrying Survivin Gene by Homologous Recombination in Bacteria

目的:研究细菌内同源重组法构建靶向Survivin的腺病毒载体及其体外扩增表达。方法:将survivin基因克隆至穿梭质粒载体中,特异性酶切后回收、连接、转化,构建负载survivin片段的重组腺病毒载体。提取重组病毒基因酶切鉴定后,包装成病毒,并扩增到所需滴度。行Western blotting鉴定,观察重组腺病毒载体在真核细胞的表达。结果:(1)重组穿梭质粒的Mlu I酶切鉴定结果显示,酶切结果均与相应的载体及目的片段的大小相符合,基因测序结果基本一致。(2)凝胶电泳产生了两条大约15 kb和8.5kb的片段,由图2可知重组腺病毒质粒酶切充分完全,且回收率较高。(3)重组腺病毒载体转染AD293细胞24 h后已出现细胞病变效应,病变细胞细胞核变大。(4)D260/OD280的值为1.92,表明重组腺病毒纯度较高。(5)AD293细胞病毒上清中有能与抗Survivin单抗反应的蛋白,其相对分子质量与理论值相吻合,阴性对照组无对应条带出现。结论:本方法成功构建表达了含Survivin的重组腺病毒载体,为进一步进行Survivin基因功能的研究提供了实验基础和理论支持。

Objective： To construct recombinant adenovirus vector carrying Survivin gene by homologous recombination in bacteria and to detect its expression in vitro. Methods： Survivin gene was cloned to adenoviral shuttle plasmid p Ad Track-CMV. Then the resultant p Ad Track-CMV- Survivin was cotransfected into E.coli.DH5α bacteria with the adenovirus back bone plasmid p Ad Easy-1. The adenovirus plasmid carrying Survivin was generated with omologous recombination in bacteria and the adenoviruses were produced in AD293 cells. AD293 cells were infected with adenoviruses and the expression of Survivin was detected by CPE and western blot.Results：（1） The enzyme digestion results coincided with the corresponding vector and target fragment size, and the gene sequencing results were also consistent.（2）The gel electrophoresis results showed a fragment of about 15 kb and a fragment of about 8 kb. The recombinant adenovirus plasmids were fully digested, and the recovery rate was high.（3）After 24 h, the recombinant adenovirus vector was transfected into AD293 cells, and cytopathic effect appeared.（4） The value of D260/OD280 is 1.92, indicating that the recombinant adenovirus was of high purity.（5） After package, anti Survivin monoclonal antibody of the virus appeared in the supernatant protein, while no bands appeared in the negative control adenovirus. Conclusion： The method successfully constructed the recombinant adenovirus vector containing Survivin, and provided an experimental basis and theoretical support for further research.