【摘要】 目的　构建人胃黏膜细胞cDNA文库 ,并选取VacA毒素的p37片段将其克隆到 pGBKT7以表达BD p37融合蛋白作为诱饵。为进一步用所此诱饵来筛选所构建文库以其找到与VacA相互作用的蛋白奠定基础。方法　用TRIzol试剂从胃腺癌病人手术切除的正常胃黏膜细胞中抽取总RNA经LD PCR得到双链cDNA ,参照Clontech的MATCHMAKERLibraryConstructionandScreeningKit构建cDNA文库并用文库所含的独立克隆数和含插入片段的克隆数占总克隆数的比例来评价酵母双杂交文库的质量。用PCR从Helicobacterpylor基因组中扩增出p 37基因将其克隆入 pGBKT7载体并使插入片段与GAL4BD同框 ,插入片段的大小和序列的正确性由双酶切和测序得以证实。结果　文库中共有 1.9× 10 6个独立克隆 ,含插入片段克隆所占比例为 91.7%。双酶切和测序结果表明 p37的正确插入 ,Westernblot结果证明了融合蛋白的表达。 结论　构建了较高质量的cDNA文库和与GAL4BD融合的诱饵蛋白 ,为进一步的研究奠定了基础。更多还原

【Abstract】 To construct the human gastric mucosa cDNA library and the recombinant plasmid pGBKT7 p37, the total RNA was isolated from normal gastric mucosa taken from surgically resected stomach of patient with gastric adenocarcinoma using TRIzol reagent under the direction of the manufacturer, and the double strand cDNA was obtained through LD PCR. Construction of cDNA library was performed by means of the direction of MATCHMAKER Library Construction and Screening Kit Clontech), and the quality of the two hybrid library was checked by estimating the number of the independent clones obtained and the percentage of library clones containing an insert. The p37 gene was amplified from Helicobacter pylori genomic DNA and cloned in frame into vector pGBKT7. It was found that there were 1.9×10 6 independent clones in the library and the percentage of insert containing clones was 91.7%. As confirmed by the result of sequencing, the p37 gene had been inserted correctly into vector pGBKT7 and the expression of the fusion protein was confirmed by Western blotting analysis. Our results indicate that a high quality yeast two hybrid cDNA library and a bait fused to GAL4 BD domain were constructed, thus providing foundation for further studies to find the proteins interacting with VacA.更多还原