细菌内同源重组快速构建和制备表达β-半乳糖苷酶的重组腺病毒

The construction of recombinant adenoviral plasmid by homologous recombination in bacteria and the preparation of recombinant adenovirus expressing β-galactosidase

目的 :利用细菌内同源重组快速构建重组腺病毒质粒和制备重组腺病毒。方法 :制备感受态BJ5 183,将pAdEasy - 1转化BJ5 183,制备含 pAdEasy - 1的BJ5 183感受态 ,将线性化的 pShuttle -CMV -LacZ转化含 pAdEasy- 1的BJ5 183感受态菌。采用细菌中同源重组法构建重组腺病毒质粒 pAdEasy - 1-LacZ。pAdEasy - 1-LacZ用PacⅠ线性化后 ,再用LipoVec介导其转染至AD2 93细胞内包装扩增出重组腺病毒颗粒 ,采用CsCl密度梯度离心法纯化重组腺病毒AdEasy - 1-LacZ。采用X - gal染色观察LipoVec介导的重组腺病毒质粒的转染效果及病毒包装情况。将纯化的AdEasy - 1-LacZ转染HVSMC ,X -gal染色观察基因转染效率及基因表达情况。 结果 :pShuttle -CMV -LacZ成功地转化了含 pAdEasy - 1的BJ5 183,并在其内发生了同源重组。X - gal染色证实了 pAdEasy - 1-LacZ转染AD2 93后 ,产生了重组腺病毒AdEasy - 1-LacZ。病毒滴度为 3.9× 10 12 pfu/ml。AdEasy - 1-LacZ转染HVSMC后 ,β -gal在HVSMC中得到了有效表达。 结论 :细菌内同源重组法是一种快速构建重组腺病毒质粒的方法 ,AdEasy - 1-LacZ的制备为基因治疗研究提供了一良好对照载体。

Objective To construct recombinant adenoviral plasmid containing β-galactosidase cDNA using homologous recombination in bacteria and to prepare recombinant adenovirus particles expressing β-galactosidase. Methods Adenoviral backbone plasmid was transformed into competent BJ5183 and the competent BJ5183 transformed with pAdEasy-1 was prepared. The linearized pShuttle-CMV-LacZ with PmeⅠdigestion and CIAP dephosphorylation was transformed into the competent BJ5183 transformed with pAdEasy-1. The identified recombinant adenoviral plasmid pAdEasy-1-LacZ was digested with PacⅠ and transfected into AD293 cells with cationic liposome LipoVec to package recombinant adenovirus particles AdEasy-1-LacZ. AdEasy-1-LacZ was amplified by repeated rounds of infection of AD293 cells with supernatant of the recombinant adenovirus. AdEasy-1-LacZ was purified with CsCl density gradient ultracentrifugation. The purified AdEasy-1-LacZ was transfected into human vascular smooth muscle cells(HVSMC). X-gal staining was performed to monitor the expression of β-galactosidase gene.Results pShuttle-CMV-LacZ was successfully transformed into competent BJ5183 transformed with pAdEasy-1 and homologous recombination between pAdEasy-1 and pShuttle-CMV-LacZ within BJ5183 bacteria took place. LipoVec transfected PacⅠ-digested pAdEasy-1-LacZ into AD293 cells. X-gal staining confirmed the packaging of recombinant adenovirus AdEasy-1-LacZ within AD293 cells and the expression of β-galactosidase in HVSMC,respectively. The viral titer was 3.9×10 12pfu/ml.Conclusion Homologous recombination between adenoviral backbone plasmid and shuttle plasmid containing exogenous gene within bacteria should expedite the process of generating recombinant adenoviruses and avoid the plaque purification. This system may provide the basis for construction of recombinant adenovirus expressing therapeutic gene of interest. AdEasy-1-LacZ is a useful tool in the research of safety, feasibility and the efficiency of gene transfer mediated by recombinant adenovirus.