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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。
Objective To construct a cDNA library of human gastric mucosal cells and select the p37 fragment of VacA toxin to clone it into pGBKT7 to express BD p37 fusion protein as bait. The library was further screened with this bait to lay the foundation for its finding of a protein that interacts with VacA. METHODS: Total RNA was extracted from normal gastric mucosal cells surgically removed from patients with gastric adenocarcinoma using TRIzol reagent. The double-stranded cDNA was obtained by LD PCR. The cDNA library was constructed according to the MATCHMAKER Library Technique and Screening Kit by Clontech. The number of independent clones contained in the library and the number of clones containing the insert The ratio of total clones was used to assess the quality of the yeast two-hybrid library. The p 37 gene was amplified by PCR from Helicobacter pylor genome and cloned into pGBKT7 vector. The inserted fragment was cloned into the frame of GAL4BD. The correct size and sequence of the inserted fragment were confirmed by double enzyme digestion and sequencing. A total of 1.9 × 10 6 independent clones were found in the library of the results. The percentage of clones containing insert was 91.7%. Double digestion and sequencing results showed that the correct insertion of p37, Western blot results confirmed the expression of the fusion protein. Conclusion A higher quality cDNA library and bait protein fused with GAL4BD were constructed, which laid the foundation for further research.