CtBP1基因shRNA真核表达载体的构建及其干扰效果的初步鉴定

Construction of shRNA eukaryotic expression vector of CtBP1 gene and preliminary identification of its interference efficiency

目的：构建C-末端结合蛋白-1（CtBP1）基因RNA干扰（RNAi）的真核表达载体,转染人子宫内膜癌耐药细胞株B-MD-C1（ADR＋/＋）,初步鉴定其干扰效果。方法：以人CtBP1基因为靶基因,以pGenesil-1质粒为载体,根据GenBank数据库提供的CtBP1基因核苷酸序列,选择设计两条siRNA干扰序列,将带9个碱基茎环结构的干扰序列插入带有绿色荧光蛋白（EGFP）基因、卡那霉素（Kanr）抗性基因及U6启动子的真核表达载体pGenesil-1中,构建针对CtBP1基因的shRNA真核表达载体,转化大肠杆菌DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析。确认载体构建成功后,用脂质体LipofectamineTM2000将重组质粒瞬时转染人子宫内膜癌耐药细胞株B-MD-C1（ADR＋/＋）,在荧光显微镜下观察绿色荧光蛋白表达,计数转染细胞数,计算转染效率,RT-PCR法检测载体对CtBP1基因的表达抑制效果。结果：构建针对CtBP1基因的pGenesil-1-CtBP1小发夹RNA（pGenesil-1-CtBP1-shRNA）,经限制性内切酶酶切、PCR和DNA测序证实与设计完全一致;在荧光显微镜下观察到B-MD-C1（ADR＋/＋）细胞表达绿色荧光蛋白（EGFP）,证实重组质粒己转染入细胞,转染效率达到60%-70%;RT-PCR结果显示B-MD-C1（ADR＋/＋）细胞中CtBP1基因被特异抑制,基因表达抑制率达40%以上,与转染试剂对照组、空白对照组和阴性序列对照组间的差异均具有统计学意义（P均〈0.05）。结论：成功构建CtBP1基因的shRNA表达载体,可以有效抑制B-MD-C1（ADR＋/＋）细胞上CtBP1基因表达。为进一步研究CtBP1基因功能进而逆转肿瘤的多药耐药奠定基础。

OBJECTIVE：To design and construct the eukaryotic expression vector of human CtBP1 gene short hairpin RNA（shRNA）then to transfect human endometrial carcinoma cells B-MD-C1（ADR＋/＋）,and to investigate the silencing effect of CtBP1 shRNA on the expression of human CtBP1 gene.METHODS：siRNAs were designed by targeting human CtBP1 gene using pGenesil-1 as vector.According to the CtBP1 cDNA sequence in GenBank,2 pairs of oligonucleotides were designed.A hairpin loop with 9 base pairs was added into the interference sequence and inserted into the eukaryotic expression vectors which contain the EGFP gene,kanr gene and U6 promoter.After primer annealing,they were inserted into plasmid pGenesil-1 to construct the shRNA eukaryotic expression vector.The recombinant plasmid were transformed into E.coli DH5α strains,and the positive strains were identified by enzyme digestion and sequence analysis.The recombinant vector were transfected into B-MD-C1（ADR＋/＋）cells with LipofectamineTM 2000.The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by reverse transcription polymerase chain reaction（RT-PCR）.RESULTS：The pGenesil-1-CtBP1 shRNA of recombinant plasmid was constructed and confirmed by DNA sequencing.Fluorescence microscope for lots of green fluorescence showed that shRNAs had transfected B-MD-C1（ADR＋/＋）cells（transfection rate was 60%～70%）.And the CtBP1 mRNA were effectively inhibited by pGenesil-1-CtBP1-shRNA of recombinant plasmid（inhibition rate was above 50%）.CONCLUSION：The eukaryoticexpression vectors of CtBP1 shRNA were constructed successfully.The shRNA could effectivelly inhibit the expression of CtBP1 gene,laiding a foundation to further study for its function and reverse the cancer multidrug resistence by blocking CtBP1 gene expression.