抑癌基因TCF21重组腺病毒载体的构建及鉴定

目的：利用大肠杆菌BJ5183细菌内同源重组法构建重组腺病毒pAd-TCF21载体。方法设计TCF21cDNA 扩增引物，从真核表达载体pCMV-SPORT6.1-TCF21中扩增TCF21的DNA序列，与腺病毒穿梭质粒pAdTrack-CMV进行连接，构成穿梭质粒pAdTrack-TCF21；然后再用经PmeI酶线性化的pAdTrack-TCF21转化含pAdeasy-1的超感受态AdBJ5183，采用细菌内同源重组法构建腺病毒质粒pAd-TCF21；筛选同源重组质粒阳性克隆，提取pAd-TCF21质粒经PacI酶切鉴定和PCR鉴定。结果线性化的pAdTrack-TCF21转化含pAdeasy-1的超感受态AdBJ5183，12~20h后获得了40%阳性重组质粒克隆，经酶切获得＞23kb的大片段和3.0 kb的特征性片段，PCR反应扩增出了540bp的片段，证明重组腺病毒质粒中已成功插入目的基因TCF21。结论细菌内同源重组法成功构建了含TCF21基因的重组腺病毒质粒，为下一步腺病毒介导的TCF21转染A549细胞的研究打下基础。

ObjectiveTo construct recombinant adenovirus plasmids by using a method of homologous recombination in bacteria with TCF21 as target gene .Method The TCF21cDNA primers were designed,the DNA sequence of TCF21 was amplified from eukaryotic vector pCMV-SPORT6.1-TCF21 and ligated into the adenovirus shuttle plasmid pAdTrack-CMV,the shuttle plasmid named pAdTrack-TCF21 was constructed. Then the pAdTrack-TCF21 was linealized with PmeI and transformed into ultracompletent BJ5183 containing pAdeasy-l,then recombinant advenovirus plasmid pAd-TCF21 was constructed by homologous recombination in bacteria.Positive clone of homologous recombination was selected,pAd-TCF21 was extracted and identified by PacI digestion and PCR.ResultsThe linealized pAdTrack-TCF21 was transformed into ultracompletent BJ5183 containing pAdeasy-1.There were over 40% positive recombinant plasmid.There were two bands：3kb and larger than 23 kb when pAd-TCF21 was digested with PacI.A 540bp TCF21 cDNA fragment was amplified by PCR.The target gene tcf21 was successfully cloned into adenovirus genomie DNA plasmid by digesting with PacI and the PCR technique .ConclusionThe recombinant adenoviral plasmid containing TCF21 was successfully constructed with homologous recombination in bacteria.This study provides a basis for the next research of the transfection of A549 cells by adenovirus-mediated TCF21.