凋亡抑制蛋白Livin shRNA真核表达载体的构建

Construction of Livin shRNA Eukaryotic Expression Vectors

目的设计构建Livin的shRNA真核表达载体,并转染宫颈癌HeLa细胞,进一步研究该表达载体对目的基因Livin的沉默效果,为宫颈癌基因治疗探索新方法。方法以Livin为靶基因,以Pgenesil-1质粒为载体,构建重组体,根据Livin基因cDNA序列,设计shRNA寡核苷酸序列,退火后克隆到空载体Pgenesil-1中,转化DH5α菌株,提取质粒,进行酶切鉴定和测序分析。将构建好的真核表达载体转染宫颈癌HeLa细胞。结果经酶切鉴定出的重组体测序结果与目的序列相同,重组载体构建成功,并被成功转入宫颈癌HeLa细胞,可见绿色荧光蛋白表达,48 h转染效率最高。结论利用RNA干扰技术可成功构建小干扰RNA重组体,为进一步研究宫颈癌基因治疗奠定基础。

Objective To construct Livin shRNA eukaryotic expression vectors to transfect into Hela cells in order to fur ther study the silencing effects of the vector on the targeting gene Livin. Methods The recombinant was constructed by using Livin as targeting gene and Pgenesil-1 as vector. According to the cDNA sequence of Livin gene, shRNA oligonucleotides were synthesized and inserted into Pgenesil-1 after annealing to generate shRNA eukaryotic expression vectors. After DHSa strains were transformed with the vectors, plasmids were extraeted, and the recombinant sequences were identified by enzyme digestion. The eukaryotic expression vectors were transfected into HeLa cells. Results The recombinant sequence identified by enzyme digestion was the same as the targeting one. The recombinant vectors were established successfully. In the HeLa cells transfected with the recombinant vectors, the expression of green fluorescent protein （GFP） was detected, and the transfection efficiency reached the peak at 48 h. Conclusion shRNA recombinant can be established successfully by RNAi technique and transfected into HeLa cells.