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目的构建人分泌的凋亡相关蛋白1(secreted apoptosis-related protein 1,SARP1)基因酵母双杂交诱饵载体,为鉴定SARP1基因相互作用蛋白、探讨SARP1基因在瘢痕组织中的生物学功能奠定基础。方法根据GenBank中SARP1的mRNA序列,设计分别带有NdeⅠ和SalⅠ酶切位点的上、下游引物,人工合成SARP1基因片段,扩增SARP1基因片段,构建含pGBKT7-SARP1基因的重组质粒,用NdeⅠ和SalⅠ进行双酶切、PCR,凝胶电泳及测序。用醋酸锂法将序列正确的重组质粒pGBKT7-SARP1(卡那霉素抗性筛选)转化入AH109酵母菌株,在缺陷型培养基上观察pGBKT7-SARP1在AH109中的表达情况。结果 SARP1基因扩增后的片段大小符合预期,并成功克隆入pGBKT7载体中;酶切后凝胶电泳及DNA测序显示pGBKT7-SARP1基因重组载体构建正确,对酵母菌株AH109无毒性,且不具自主激活报告基因的效应。结论成功构建了pGBKT7-SARP1基因酵母双杂交诱饵载体。
Objective To construct a yeast two - hybrid bait vector containing secreted apoptosis - related protein 1 (SARP1) gene and lay a foundation for the identification of the SARP1 - interacting protein and the biological function of SARP1 in scar tissue. Methods According to the mRNA sequence of SARP1 in GenBank, the upstream and downstream primers with NdeⅠand SalⅠ restriction sites were designed respectively to synthesize the SARP1 gene fragment and amplify the SARP1 gene fragment. The recombinant plasmid containing pGBKT7-SARP1 gene was constructed and expressed with NdeⅠ And Sal Ⅰ double digestion, PCR, gel electrophoresis and sequencing. The recombinant plasmid pGBKT7-SARP1 (kanamycin resistance screening) with the correct sequence was transformed into AH109 yeast strain by lithium acetate method, and the expression of pGBKT7-SARP1 in AH109 was observed on the deficient medium. Results The size of amplified fragment of SARP1 gene was in line with expectation and was successfully cloned into pGBKT7 vector. After digestion with gel electrophoresis and DNA sequencing, the recombinant plasmid pGBKT7-SARP1 was constructed correctly and non-toxic to yeast strain AH109 Report the effect of the gene. Conclusion The yeast two-hybrid bait vector pGBKT7-SARP1 was successfully constructed.