LSPA1早期基因gp22细菌双杂交诱饵载体与筛选文库的建立

Construction of LSPA1 Early Gene gp22 Bacterial Two-hybrid Bait Vector and Screening Library

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。

This study aims to construct a bait vector pKT25-gp22 and the gene library of Salmonella paratyphi A in bacterial two-hybrid system for further screening study. The gp22 gene was obtained by PCR amplification and then cloned into pKT25 to constitute the bait plasmid pKT25-gp22. The genomic DNA of S. paratyphi A was extracted and partially digested with Sau3A I . The fragments between 250 tol 500 bp were re-natured and ligated to BamH I site of pUT18C plasmid, and the genomic DNA library was obtained. The bait plasmid pKT25-gp22 and pUT18C were co-transformed to BTH101, and the self-activation of bait plasmid pKT25-gp22 and the toxicity of bait protein to the host bacteria were detected. The library plasmids were transformed to JM109 competence and the library diversity was identified by PCR. The results revealed that the bait vector was successfully constructed without self-activation of the transcription of reporter gene and non-toxic to the growth of bacteria. The genomic library covering 8 times of S. paratyphi A genome met the requirements of screening. In conclusion, the bait plasmid pKT25-gp22 and the genomic DNA library of S. paratyphi A were successfully constructed, which can be utilized in bacterial two-hybrid study.