细菌内同源重组高效制备含绿色荧光蛋白和抗凋亡基因bcl-XL的重组腺病毒载体

目的:制备含绿色荧光蛋白和抗凋亡基因bcl-X_L的重组腺病毒载体。方法:采用PCR方法从质粒pEGFP-C3-bcl-X_L中扩增出bcl-X_L基因,再亚克隆至腺病毒穿梭质粒中,形成转移质粒pAdTrack-CMV-bcl-X_L;然后与pAdEasy-1共转化BJ5183菌,采用细菌内同源重组法构建重组腺病毒质粒pAdEasy-1-gfp-bcl-X_L,筛选同源重组的阳性克隆;抽提的pAdEasy-1-gfp-bcl-X_L经PacI线性化后,再用LIPOFECTAMINE^TM2000介导转染至人胚肾293细胞内包装扩增出重组腺病毒颗粒;采用PCR方法对重组腺病毒进行鉴定;用氯化铯超高速梯度离心纯化获取高滴度的重组腺病毒rAd-gfp-bcl-X_L。结果:由pAdTrack-CMV-bcl-X_L和pAdEasy-1共转化BJ5183菌16-20h后,可获得35%的阳性重组体细菌克隆。由重组质粒DNA经293细胞包装后产生的重组腺病毒,经PCR检测表明含有目的基因bcl-X_L。氯化铯纯化所得重组腺病毒滴度约为65×10¹²PFU/L。结论:用细菌内同源重组法可以高效、简便、快捷地制备出重组腺病毒质粒载体,重组体病毒质粒经293细胞包装、扩增和氯化铯纯化后可制备出高滴度的重组腺病毒颗粒rAd-gfp-bcl-X_L,为人类相关疾病的基因治疗提供良好的基因转染载体。

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.