应用酵母双杂交筛选人胃癌耐药细胞SIVA-1基因相互作用蛋白

Screening of SIVA-1 gene interacting proteins in drug-resistant human gastric cancer cells by yeast two hybrid

目的:应用酵母双杂交筛选人胃癌耐药细胞中与SIVA-1基因相互作用的蛋白。方法:构建人胃癌耐药细胞的cDNA文库,运用酵母双杂交及体外免疫共沉淀实验方法筛选SIVA-1相关作用蛋白,探讨其对胃癌耐药细胞耐药性的影响机制。结果:成功构建人胃癌耐药细胞cDNA文库,滴度为4.78×10^(6)cfu/mL,重组率>95%,扩增后滴度达9.50×10^(9)pfu/mL,成功构建诱饵表达载体pGBKT7-SIVA-1,且经自激活检测表明重组诱饵载体pGBKT7-SIVA-1不具有自激活性。经过初筛,我们获得能同时激活三个报告基因(Ade2、His3、LacZ)的阳性克隆21个,随后将21个阳性克隆进行质粒抽提、扩增并进行酵母回转实验,最终获得4个与SIVA-1截短体相互作用的阳性候选克隆。对这四个文库插入片段进行基因测序,最终得到2个不同的与SIVA-1相互作用的蛋白:UXT(泛表达转录子)和PCBP1(多聚胞嘧啶结合蛋白1)。经体外免疫共沉淀实验后,最终筛选出与胃癌耐药细胞SIVA-1相互作用的PCBP1。结论:PCBP1与SIVA-1之间存在蛋白相互作用关系。

Objective:To screen the proteins interacting with SIVA-1 gene in drug-resistant human gastric cancer cells by yeast two hybrid technique.Methods:The cDNA library of human gastric cancer drug-resistant cells was constructed, and the related proteins of SIVA-1 were screened by yeast two hybrid and in vitro immunoprecipitation assay, so as to explore the mechanism of its effect on the drug resistance of gastric cancer drug-resistant cells.Results:The cDNA library of drug-resistant human gastric cancer cells was successfully constructed, and the titer was 4.78×10^(6) cfu/mL,recombination rate>95%,and the titer was 9.50×10^(9) pfu/mL after amplification.The bait expression vector pGBKT7-SIVA-1 was successfully constructed, and the self activation test showed that the recombinant bait vector pGBKT7-SIVA-1 did not have self-excited activity.After preliminary screening, we obtained 21 positive clones that can activate three reporter genes(Ade2,His3 and LacZ) at the same time.The 21 positive clones were further extracted for plasmid, amplified and yeast rotation experiment was performed.Finally, four positive candidate clones interacting with SIVA-1 truncate were obtained.After gene sequencing of the four library inserts, two different proteins interacting with SIVA-1 were obtained: UXT(ubiquitin transcript) and PCBP1(polycytosine binding protein 1).After in vitro immunoprecipitation experiment, PCBP1 interacting with gastric cancer drug-resistant cell SIVA-1 was finally screened.Conclusion:There is a protein-protein interaction between PCBP1 and SIVA-1.