三叶因子2相互作用蛋白基因在胃癌细胞cDNA文库中的筛选

Identification of genes encoding human trefoil factor 2-interacting proteins by screening a cDNA library of gastric cancer cells

目的:筛选并克隆人胃癌细胞cDNA文库中与三叶因子2(trefoil factor family2,TFF2)蛋白相互作用的蛋白基因.方法:RT-PCR方法验证胃癌细胞存在TFF2mRNA表达;PCR法扩增TFF2基因,NotⅠ与SalⅠ双酶切后定向克隆到酵母表达载体pDEST32,构建TFF2的诱饵质粒;Western blot验证TFF2诱饵质粒在酵母细胞中相应融合蛋白的表达;将诱饵质粒和人胃癌-cDNA文库猎物质粒共同转化MaV203酵母感受态细胞,通过营养缺陷型培养基(Ura、His)与X-gal进行3重筛选阳性克隆,提取阳性克隆质粒测序,应用蛋白质数据库及生物信息学技术,对测序结果进行分析.结果:RT-PCR法证实胃癌细胞中存在TFF2mRNA表达;构建pDEST32-TFF2诱饵表达质粒成功;Western blot法证实诱饵质粒转化酵母细胞后正确表达TFF2-GAL4DBD融合蛋白;通过酵母双杂交筛选胃癌细胞cDNA文库共获得35个表达His、Ura及X-gal报告基因的阳性克隆,其中测序成功为16个克隆,包含12个已知蛋白基因与4个未知功能基因.结论:从胃癌细胞cDNA文库中筛选出多种TFF2相互作用蛋白基因,其可能与胃癌的发生发展密切相关.

AIM：To identify and clone genes encoding human trefoil factor 2 （TFF2）-interacting proteins by screening a cDNA library of gastric cancer cells. METHODS：The expression of TFF2 mRNA in gastric cancer was verified by reverse transcription-polymerase chain reaction （RT-PCR）. The TFF2 gene was amplified by PCR,double digested with Not I and Sal I,and cloned into yeast expression vector pDEST32 to construct TFF2 bait plasmid. The expression of TFF2-GAL4DBD fusion protein by the bait plasmid in yeast cells was confirmed by Western blot. The TFF2 bait plasmid and a prey plasmid （containing a cDNA library of human gastric cancer cells） were cotransfected into Mav203 competent yeast cells. The positive clones were isolated using selective medium and sequenced. The sequenced genes were then compared with sequences deposited in the GenBank database. RESULTS：RT-PCR analysis proved that TFF2 mRNA was expressed in gastric cancer. The pD-EST32-TFF2 bait plasmid was successfully constructed. Western blot analysis confirmed that the TFF2 bait plasmid could correctly express the TFF2-GAL4DBD fusion protein. A total of 35 positive clones were obtained from the yeast two-hybrid screening. Sixteen positive clones were successfully sequenced,of which 12 represented known genes and 4 unknown genes. CONCLUSION：Multiple genes encoding TFF2-interacting proteins were identified from the cDNA library of gastric cancer cells. These genes may be closely associated with the development and progression of gastric cancer.