青霉素结合蛋白PBP1a的克隆表达和对肺炎链球菌耐药性的影响  

Cloning and expression of penicillin-binding protein PBP1a and its effect on the penicillin resistance of Streptococcus pneumonia

作　　者：潘玲[1] 周侠[2] 杨致邦[2] 邓西川[2] 

机构地区：[1]重庆市肿瘤研究所,重庆400030 [2]重庆医科大学基础医学实验教学中心病原生物学与免疫学实验室,重庆400016

出　　处：《中国病原生物学杂志》2012年第12期901-904,923,共5页Journal of Pathogen Biology

摘　　要：目的克隆并表达肺炎链球菌青霉素结合蛋白PBP1a,探讨其对青霉素耐药的影响。方法化学合成肺炎链球菌含STMK区的pbp1a基因片段,PCR扩增后进行1.0%琼脂糖凝胶电泳鉴定。将扩增的pbp1a基因连接pUC19载体,构建重组质粒pUC19-pbp1a,转化DH5α,PCR扩增后进行1.5%琼脂糖电泳分析和DNA测序鉴定。将pUC19-pbp1a转化对青霉素敏感、中度敏感和耐药,并存在不同的pbp1a、pbp2b突变的肺炎链球菌,SDS-PAGE分析PBP1a的表达,并测定青霉素对转化菌的MIC。结果 pbp1a基因扩增产物经琼脂糖凝胶电泳为330bp,大小与预期一致。重组质粒基因全长为360bp,扩增产物约330bp,测序无碱基缺失、插入等突变。重组质粒转化存在不同的pbp1a、pbp2b突变和对青霉素不同敏感性的肺炎链球菌后,大量表达约110ku蛋白,其中对青霉素敏感和中度敏感的无pbp1a突变株MIC无变化,对中度敏感菌中的pbp1a突变株MIC略有变化,对存在pbp1a突变的耐药菌株MIC显著降低(P<0.01)。结论在肺炎链球菌中成功表达了青霉素结合蛋白PBP1a,可使其对青霉素的耐药性显著降低。Objective To determine the effect of PBP1a on the resistance of Streptococcus pneumonia to penicillin.The pbp1a gene coding for a penicillin-binding protein in S.pneumoniae was cloned and expressed.Methods The pbp1a fragment containing the STMK region from S.pneumoniae was chemically synthesized and amplified with PCR.The amplified products were identified by 1.5% agarose electrophoretic analysis.The amplified pbp1a was ligated with a pUC19 vector.The recombinant pUC19-pbp1a plasmid was converted and translated into DH5α.The pbp1a gene in DH5α was amplified and identified by 1.5% agarose electrophoretic analysis again and then subjected to DNA sequencing analysis.Then the pUC19-pbp1a was transformed into S.pneumoniae with sensitivity,intermediate sensitivity,and resistance to penicillin and with various mutations of pbp1a and pbp2b.The expression of PBP1a was analyzed by SDS-PAGE and the MIC of penicillin for the transformed S.pneumoniae was detected.Results A band of about 330 bp was observed in agarose electrophoretic analysis after pbp1a was amplified with PCR,and this size coincided with expectations.The recombinant plasmid was 360 bp in length,and the sequences of the recombinant plasmid lacked mutations such as missing or inserted bases.The MIC of penicillin for strains with sensitivity and intermediate sensitivity to penicillin and with no pbp1a mutation did not change.The MIC of penicillin for strains with intermediate sensitivity and pbp1a mutation decreased slightly.The MIC for strains with resistance to penicillin and pbp1a mutation decreased markedly.There were significant differences between transformed and untransformed S.pneumoniae（P〈0.01）.Conclusion The penicillin-binding protein PBP1a of S.pneumoniae was successfully expressed and markedly decreased the resistance of S.pneumoniae to penicillin.

关 键 词：肺炎链球菌 PBP1a 克隆表达 耐药性 

分 类 号：R378.12[医药卫生—病原生物学]