MafA基因重组腺病毒的构建及其对小鼠肝癌细胞的影响

目的利用AdEasy-1系统构建胰岛素转录因子之一的MafA基因重组腺病毒（Ad-MafA）,并观察其对小鼠肝癌细胞的影响。方法将质粒cDNA3/MafA扩增、酶切获得MafADNA片段插入腺病毒穿梭载体质粒pAdTrack-CMV的巨细胞病毒（CMV）启动子下游,构建重组穿梭载体pAdTrack-CMV-MafA,线性化后与骨架载体AdEasy-1在细菌BJ5183内同源重组得到腺病毒质粒pAd-MafA,经293细胞包装后得到含MafA全长DNA片段的重组腺病毒AdMafA;将Ad-MafA体外感染小鼠肝癌细胞（Hepa1-6）,以RT-PCR和Westernblot检测MafA在hepa1-6细胞的表达。结果连接、重组后通过酶切法筛选出pAd-MafA,经293细胞包装,5d后观察到绿色荧光蛋白（GFP）明显表达,通过反复感染HEK细胞扩增得到高滴度的重组腺病毒,Ad-MafA体外感染肝癌细胞1d后,MafA基因表达和蛋白表达明显增加。结论利用新型腺病毒载体AdEasy-1系统可在短期内制备同时表达GFP和MafA的重组腺病毒（Ad-MafA）,Ad-MafA体外感染小鼠肝癌细胞可显著提高MafA的表达,这将为基因治疗糖尿病提供新的手段。

Construction of recombinant adenoviruses carrying MafA and its effect on hepatic carcinoma cells

Objective To construct the recombinant adenoviruses- Ad- MafA inserted both mouse MafA and green fluorescent protein （GFP） cDNA drived by CMV promoter using homologous recombination in bacteria provided by AdEasy system and to investigate the effect of Ad - MafA on mouse hepatic carcinoma cells. Methods The MafA cDNA was obtained from the plasmids - cDNA3/MafA by digestion,and the shuttle plasmid- pAdTrack- CMV- MafA,which the MafA cDNA was inserted into the downstream of CMV promoter, was established by ligation. Then the linearized shuttle plasmid was co -transformed into bacteria with backbone vector AdEasy - 1 to obtain the recombinant adenoviral plasmids - pAd - MafA by homologous recombination. After packed in 293 cells, the recombinant adenoviruses - Ad - MafA were generated. The expression of MafA in mouse hepatic carcinoma cell was detected by RT - PCR and western blot. Results The recombinant plasmid pAd- MafA was established by homologous recombination and confirmed by restriction endonnclease digestion and sequencing. GFP expression could be observed on the fifth day after packing of the linearized pAd - MafA in 293 cells and 4 × 10^10 efu/mL titer of Ad - MafA was obtained by CsCl gradient purification. When the mouse hepatic carcinoma cells were infected by the viruses for 1 day, the gene and protein expression of MafA in cells increased significantly. Conclusions MafA can be simply and rapidly generated by using the AdEasy system. The infection of mouse hepatic carcinoma cells by Ad - MafA can result in the high expression of MafA, Ad - MafA may serve as a new tool for gene therapy of diabetes.