细菌内同源重组快速构建和制备表达hSDF-1α的重组腺病毒

Construction of recombinant adeno-viral plasmid bearing hSDF-1α cDNA by homologous recombination in bac-teria and preparation of recombinant adenovirus expressing hSDF-1α

目的:利用细菌内同源重组快速构建携带hSDF-1αcDNA重组腺病毒质粒和制备表达hSDF-1α重组腺病毒.方法:制备感受态BJ5183,将pAdEasy-1转化BJ5183,制备含pAdEasy-1的BJ5183感受态,将线性化的pShuttle-EGFP-hS-DF-1α转化含pAdEasy-1的BJ5183感受态菌.采用细菌中同源重组法构建重组腺病毒质粒pAd-EGFP-hSDF-1α.pAd-EG-FP-hSDF-1α用PacI线性化后,再用LipoVec介导其转染至AD293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Ad-EGFP-hSDF-1α.采用荧光显微镜下观察LipoVec介导的重组腺病毒质粒的转染效果及病毒包装情况.结果:pShuttle-EGFP-hSDF-1α成功地转化了含pAdEasy-1的BJ5183,并在其内发生了同源重组.荧光显微镜下观察证实了pAd-EGFP-hSDF-1α转染AD293后,产生了重组腺病毒Ad-EGFP-hSDF-1α.病毒滴度为4.1×1015pfu/L.结论:用细菌内同源重组法可快速、高效制备表达hSDF-1α的高滴度重组腺病毒.为研究hSDF-1α在动员骨髓源干细胞迁移到组织损伤部位修复组织损伤的研究奠定了基础.

AIM： To construct recombinant adenoviral plasmid containing hSDF-1α cDNA using homologous recombination in bacteria and to prepare recombinant adenovirus expressing hSDF- 1α. METHODS： Adenoviral backbone plasmid was transformed into competent BJ5183 and the competent BJ5183 transformed with pAdEasy-1 was prepared. The linearized pShuttle-EGFP- hSDF-1α plasmid with Pme I digestion and CIAP dephosphorylation was transformed into the competent cells BJ5183 transformed with pAdEasy-1. The identified recombinant adenoviral plasmid pAd-EGFP-hSDF-let was digested with Pac I and transfeeted into AD293 cells with cationic liposome LipoVee to package recombinant adenoviras Ad-EGFP-hSDF-1. Ad-EGFP-hSDF-1α was propagated by repeated rounds of infection of AD293 cells with supematant of the recombinant adenovirus. Ad-EGFP-hSDF-let was purified with CsCl density gradient ultraeentrifugation. RESULTS： pShuttle-EGFP-hSDF-1α was successfully transformed into competent BJ5183 transformed with pAdEasy-1 and homologous recombination between pAdEasy-1 and pShuttle- EGFP-hSDF-1α took place within BJ5183 bacteria. Liposome- mediated transfection of pAd-EGFP-hSDF-1α digested with Pac I into AD293 cells was performed. The packaging of recombinant adenovirus Ad-EGFP-hSDF-1α within AD293 cells was confirmed by fluorescent microscopy. The viral titer was 4.1 - 10^15 pfu/L. CONCLUSION： The recombinant adenovirus expressing hSDF- 1α was prepared successfully by a simple and rapid homologous recombination in bacteria. This study provides a basis for exploring the migration mechanism of bone marrow-derived stem cells into injured tissue such as infarcted myocardium.