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目的 :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。方法 :采用PCR方法从质粒pEGFP -C3 -bcl-XL 中扩增出bcl-XL 基因 ,再亚克隆至腺病毒穿梭质粒中 ,形成转移质粒pAdTrack -CMV -bcl-XL;然后与pAdEasy - 1共转化BJ51 83菌 ,采用细菌内同源重组法构建重组腺病毒质粒pAdEasy - 1 -gfp -bcl-XL,筛选同源重组的阳性克隆 ;抽提的pAdEasy - 1 -gfp -bcl-XL 经PacI线性化后 ,再用LIPOFECTAMINETM2 0 0 0介导转染至人胚肾 2 93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-XL。结果 :由pAdTrack -CMV -bcl-XL 和pAdEasy - 1共转化BJ51 83菌1 6 - 2 0h后 ,可获得 35 %的阳性重组体细菌克隆。由重组质粒DNA经 2 93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-XL。氯化铯纯化所得重组腺病毒滴度约为 6 5× 1 0 1 2 PFU/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 2 93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-XL,为人类相关疾病的基因治疗提供良好的基因转染载体。
Objective: To prepare a recombinant adenovirus vector containing green fluorescent protein and anti-apoptotic gene bcl-XL. Methods: The bcl-XL gene was amplified from the plasmid pEGFP-C3-bcl-XL by PCR and then subcloned into the adenovirus shuttle plasmid to form the transfer plasmid pAdTrack-CMV-bcl-XL. The recombinant adenovirus plasmid pAdEasy - 1 -gfp -bcl-XL was constructed by homologous recombination in bacteria and the homologous recombination positive clones were screened. The extracted pAdEasy - 1 - gfp - bcl-XL was analyzed by PacI linearity The recombinant adenovirus particles were then amplified by LIPOFECTAMINETM2006-mediated transfection into human embryonic kidney 293 cells. The recombinant adenovirus was identified by PCR and purified by cesium chloride ultracentrifugation gradient centrifugation High-titer recombinant adenovirus rAd -gfp -bcl-XL. Results: 35% positive recombinant bacterial clones were obtained after cotransfection of BJ51 83 bacteria by pAdTrack-CMV-bcl-XL and pAdEasy - 1 for 1-6 h. Recombinant plasmid DNA by 293 cells produced recombinant adenovirus, PCR detection showed that the target gene bcl-XL. The titer of recombinant adenovirus purified by cesium chloride was about 6 5 × 10 12 PFU / L. CONCLUSION: Recombinant adenovirus plasmid vector can be prepared efficiently, easily and rapidly by bacterial homologous recombination method. Recombinant virus plasmids can be prepared by high-titer recombination after being packaged by 293 cells, amplified and purified with cesium chloride The adenoviral particle rAd -gfp -bcl-XL provides a good gene transfer vector for the gene therapy of human-related diseases.