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目的通过建立细菌双杂交技术,筛选MRSA中与PBP2a相互作用的蛋白。方法利用PCR扩增,获得PBP2a蛋白转肽酶活性区(TPase)的编码基因,插入pRBR构建成诱饵质粒pBR-PBP2a。提取MRSA N315株基因组DNA,经Sau3AⅠ部分酶切后连接到pRAC质粒的BamHⅠ位点,获得基因组DNA表达文库;将文库质粒转化入含诱饵质粒pBR-PBP2a的报告菌株KS1,利用细菌双杂交技术进行筛选,获得与诱饵质粒编码的融合蛋白相互作用的猎物,对猎物中编码序列进行DNA测序和生物信息学分析,确定与PBP2a发生相互作用的蛋白质或多肽。结果成功构建了pBR-PBP2a诱饵质粒,可表达PBP2a TPase与大肠埃希菌RNA聚合酶α亚单位N端序列的融合蛋白。所构建的MRSA N315株基因组文库覆盖率达9倍,满足文库筛选的需要。将文库转化含诱饵质粒和报告基因的KS1宿主菌,利用细菌双杂交技术经3次筛选,共获得9个猎物克隆,其报告基因的活性均升高2倍以上,对9个猎物质粒进行了测序,信息学分析表明它们均来自于MRSA N315株基因组,插入片段最长者648 bp,最短者334 bp,9个插入片段中含14个编码基因,其中10个功能未知,1个编码二氢吡啶二羧酸合酶、1个编码二氢吡啶二羧酸还原酶,2个编码染色体解离稳定(SMC)蛋白。9个克隆中介导与PBP2a相互作用的多肽由14～46个氨基酸组成。结论利用细菌双杂交技术从MRSA基因组文库中成功筛选到与PBP2a相互作用的多肽。
OBJECTIVE To screen the proteins that interact with PBP2a in MRSA through the establishment of bacterial two-hybrid technique. Methods The gene encoding the transpeptidase activity domain (PBP2a) of PBP2a was amplified by PCR and inserted into pRBR to construct bait plasmid pBR-PBP2a. The genomic DNA of MRSA N315 strain was extracted and partially digested with Sau3A I and ligated into the BamHI site of pRAC plasmid to obtain a genomic DNA expression library. The library plasmid was transformed into the reporter strain KS1 harboring the bait plasmid pBR-PBP2a, and the bacterial two-hybrid technique was used Screening, obtaining the prey interacting with the fusion protein encoded by the bait plasmid, performing DNA sequencing and bioinformatics analysis on the coding sequence in the prey, and identifying the protein or polypeptide interacting with PBP2a. Results The bait plasmid pBR-PBP2a was successfully constructed and expressed the fusion protein of PBP2a TPase and N-terminal sequence of RNA polymerase α subunit of Escherichia coli. The constructed MRSA N315 strain genomic library coverage rate of 9 times, to meet the needs of library screening. The library was transformed with bait plasmid and reporter gene KS1 host bacteria, using bacterial two-hybrid screening technology three times, a total of nine prey clones, the reporter gene activity were increased more than 2 times, nine prey plasmid Sequence analysis and informatics analysis indicated that the two genes were all from the genome of MRSA N315. The longest insert was 648 bp, the shortest was 334 bp. The nine inserts contained 14 coding genes, of which 10 were unknown, Dipicolinate synthase, one encoding dihydrodipicolinate reductase and two encoding chromosomal dissociation stable (SMC) proteins. Nine clones mediate the interaction of PBP2a with peptides consisting of 14 to 46 amino acids. Conclusion The bacterial two-hybrid technique was successfully used to screen the polypeptides interacting with PBP2a from the MRSA genome library.