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构建细菌双杂交系统中的诱饵载体p KT25-gp22和甲型副伤寒沙门氏菌基因文库以便后续的筛选实验。PCR扩增获得gp22基因,插入p KT25构成诱饵质粒p KT25-gp22。提取甲型副伤寒沙门氏菌基因组DNA,经Sau3AⅠ部分酶切后连接到p UT18C质粒的Bam HⅠ位点,获得基因组DNA表达文库。用化转的方法将诱饵质粒与p UT18C共转入BTH101,检测诱饵质粒自激活作用,并检测诱饵蛋白对宿主菌的毒性。将文库质粒电转至JM109感受态,PCR鉴定文库的多样性。诱饵载体p KT25-gp22无自激活报告基因的能力且对细菌的生长无毒性。所构建的副甲基因组文库覆盖基因组达8倍,满足文库筛选需要。成功构建细菌双杂交系统中诱饵载体p KT25-gp22,筛选文库质量良好,可应用于细菌双杂交实验。
The bait vector p KT25-gp22 and the Salmonella paratyphi A gene library were constructed for bacterial two-hybrid system for subsequent screening experiments. The gp22 gene was amplified by PCR and inserted into pKT25 to construct bait plasmid pKT25-gp22. The genomic DNA of Salmonella paratyphi A was extracted and partially digested with Sau3AⅠ and ligated into the Bam HⅠ site of pUT18C plasmid to obtain a genomic DNA expression library. The bait plasmid and pUT18C were co-transfected into BTH101 by the transformation method to test the self-activation of the bait plasmid and to test the toxicity of the bait protein to the host bacteria. The library plasmids were electroporated into JM109 competent cells and PCR was used to identify the diversity of the libraries. The bait vector pKT25-gp22 has no ability to self-activate the reporter gene and is non-toxic to bacterial growth. The constructed parathyroid library covered 8 times the genome to meet the screening needs of the library. The bait vector p KT25-gp22 was successfully constructed in the bacterial two-hybrid system. The screening library is of good quality and can be used in bacterial two-hybrid experiments.