含人基质金属蛋白酶组织抑制因子-1重组腺病毒的构建与体外表达

Construction of Recombinant Adenovirus Vector Carrying Human TIMP-1 cDNA and Its Expression In Vitro

从人肝癌组织中提取总RNA,RT-PCR合成hTIMP-1的全长cDNA,克隆到腺病毒载体AdEasy系统的穿梭质粒pAdTrack-CMV上,与骨架质粒pAdEasy-1在BJ5183受体菌中进行同源重组,成功构建含hTIMP-1全长cDNA的重组腺病毒载体,经293细胞的包装、扩增,生成含hTIMP-1基因的重组腺病毒AdhTIMP-1并实现体外表达,为进一步研究肝癌浸润和转移机理以及肝癌的基因治疗提供实验基础。

The full-length eDNA of hTIMP-1 was obtained from a surgical patient with HCC by the method of RT-PCR. Then it was cloned into the adenoviral shuttle plasmid pAdTrack-CMV, and subsequently cotransformed into competent BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1. Thereupon, a recombinant adenoviral plasmid containing full-length eDNA of hTIMP-1 was generated by homologous recombination in E, coll. The adenoviruses （AdhTIMP-1） were packaged and amplified in adenoviral packaging cells HEK 293. Then the viral titer was checked by green fluorescent protein （GFP）, and the expression of hTIMP-1 was detected by the techniques of Western blot and RT-PCR. The recombinant adenovirus vector carrying human TIMP-1 was successfully constructed and expressed in vitro and may pave the way for further application in liver gene therapy.