耐甲氧西林金黄色葡萄球菌PBP2a相互作用蛋白的筛选

Screening of proteins interacting with PBP2a in methicillin-resistant Staphylococcus aureus

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 further understand the mechanism of PBP2a mediated the drug-resistance of methicillin-resistant Staphylococcus aureus（MRSA） with a bacterial two hybrid system.Methods The fragment encoded PBP2a TPase domain of MRSA strain N315 was amplified by PCR and then cloned into pRBR vector to construct the bait plasmid pBR-PBP2a.The genomic DNA of MRSA strain N315 was partially digested with Sau3AⅠ and the fragments between 500 to 1 500 bp were recovered,then ligated to pRAC vector prior cut with BamHⅠ to get a genomic DNA library.The bacterial two hybrid system was constructed by transforming the plasmids of the DNA library into KS1 host cells（harboring a LacZ reporter gene） prior transformed with the bait plasmid pBR-PBP2a and the prey plasmids were screened out by LacZ test.After isolation and DNA sequencing of the prey plasmids,the inserts were characterized by bioinformatic analysis and the peptide that interacts with PBP2a were obtained.Results The genomic DNA library of MRSA strain N315 and the bait plasmid pBR-PBP2a were successfully constructed.Nine clones were obtained with more than 2 times raised for LacZ activity in the bacterial two hybrid assay and their insert sequences were qualified by DNA sequencing.The longest insert was 648 bp and the shortest one was 334 bp.There were 14 genes related with the 9 inserts and 10 of them encoded proteins with unknown functions.Among the 4 familiar genes,one encoded dihydrodipicolinate synthase,one encoded dihydrodipicolinate reductase and the others encoded chromosome segregation SMC proteins.The peptides that may interact with PBP2a in the 9 prey plasmids composed of 14 to 46 amino acids.Conclusion The proteins interact with PBP2a are successfully screened by the bacterial two-hybrid system assay,which facilitates further understanding the mechanism of PBP2a mediated the drug-resistance in MRSA.