细菌内同源重组构建pAdeasy-1/hp27mt腺病毒质粒

Construction of Recombinant Adenovirus Plasmid Containing hp27mt By Homologous Recombination in Bacteria

目的 :采用细菌内同源重组法高效制备含hp2 7mt基因的重组腺病毒质粒。 方法 :hp2 7mt自载体pORF9-hp2 7mt中切出 ,亚克隆至腺病毒穿梭质粒 pShuttle -CMV中 ,构成穿梭质粒 pShuttle -CMV/hp2 7mt;然后再用经PmeI酶切的 pshuttle-CMV/hp2 7mt转化含 pAdeasy - 1的超感受态BJ51 83 ,采用细菌内同源重组法构建腺病毒质粒pAdeasy - 1 /hp2 7mt;筛选同源重组质粒阳性克隆 ,提取pAdeasy - 1 /hp2 7mt质粒经PacⅠ酶切鉴定和PCR鉴定。结果 :线性化的pShuttle-CMV/hp2 7mt转化含pAdeasy - 1的超感受态BJ51 83 ,1 2 - 2 0h后获得了30 %阳性重组质粒克隆 ,经酶切获得一大于 2 0kb的大片段和 3 .0kb的特征性片段 ,PCR反应扩增出了 2 75bp的片段 ,证明重组腺病毒质粒中已成功插入目的基因hp2 7mt。 结论 :用细菌内同源重组法可制备含hp2 7mt的重组腺病毒质粒且经济高效、简便、快捷。为转染 2

Objective To prepare recombinant adenovirus plasmids by using a novel and high efficient method of homologous recombination in bacteria with hp27mt as target gene.Methods hp27mt cDNA was digested from plasmid of pORF9-hp27mt and subcloned into plasmid of pBluescript Ⅱsk(+) .This recombinant plasmid was named after pBluescript/hp27mt . hp27mt cDNA was digested from plasmid of pBluescript / hp27mt and subcloned into pShuttle-CMV. The recombinant plasmid was named after pShutle-CMV / hp27mt ; Adenovirus genomic DNA plasmid of pAdeasy-1 was transformed into BJ5183 bacteria and prepared ultracompletent BJ5183 containing pAdeasy 1. pShuttle CMV/hp27mt was linealized with PmeI and transformed into ultracompletent BJ5183 containing pAdeasy 1, positive clone of homologous recombination was selected; the identification of recombinant adenoviral plasmid was performed with digesting with PacI and PCR. Results The linealized pShutlle CMV/hp27mt was transformed into ultracompletent BJ5183 containing pAdeasy-1. There were over 30% positive recombinant plasmid. The target gene hp27mt was successfully cloned into adenovirus genomic DNA plasmid by digesting with PacI and the PCR technique. Conclusion The method of homologous recombination in bacteria could prepare recombinant adenovirus plasmids containing hp27mt. It may provide high quality recombinant adenovirus plasmid for preparing high titer recombinant adenovirus.