【摘要】 目的:构建肺炎链球菌调控感受态形成的二元信号传导系统(TCS)基因comD/comE/comC的原核表达系统,探讨ComD和ComC封闭后细菌耐药性变化。方法:采用PCR扩增全长comD、comE和comC基因,并用常规基因工程技术建立其原核表达系统。采用SDS-PAGE及Bio-Rad凝胶图像分析系统检测目的重组蛋白rComD、rComE和rComC表达量。免疫家兔制备rComD、rComE和rComC抗血清。检测肺炎链球菌菌株ComD和ComC被抗血清封闭后对青霉素和头孢胺噻耐药性的变化。结果:与报道序列比较,所克隆的comD、comE和comC基因核苷酸和氨基酸序列相似性分别为98.4%~99.3%和99.1%~100%。所构建的工程菌E.coli BL21DE3pET42a-comD、E.coliBL21DE3pET42a-comE和E.coli BL21DE3pET32a-comC能有效表达目的重组蛋白rComD、rComE和rComC,其产量分别为细菌总蛋白的28%、25%和35%。rComD、rComE和rComC抗血清免疫双扩散效价分别为1∶4、1∶4和1∶8。ComD和/或ComC被抗血清封闭后,头孢胺噻敏感菌株可出现耐药性,但耐药菌株耐药性无明显改变,也不影响各菌株对青霉素的耐药性。结论:本研究成功地构建了肺炎链球菌comD/come/comC基因原核表达系统,为深入研究感受态形成相关TCS与耐药性关系奠定了基础。ComD和ComC与肺炎链球菌对头孢胺噻耐药性有关。更多还原

【Abstract】 Objective:To construct prokaryotic expression systems of TCS genes comD/comE/comC of Streptococcus pneumoniae,and to determine the correlation of ComD and ComC with the drug resistance.Methods: The entire comD,comE and comC genes were amplified by PCR and their prokaryotic expression systems were established by routine genetic engineering technique.SDS-PAGE and Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rComD,rComE and rComC.Rabbits were immunized with these recombinant proteins to prepare antisera.The resistance of S.pneumoniae strains to penicillin and cefotaxime was examined after ComD and ComC were blocked by antisera.Results: Compared with the reported sequences,similarities of nucleotide and amino acid sequences of the cloned comD,comE and comC genes were 98.4%~99.3% and 99.1%~100%,respectively.The constructed engineering bacteria E.coli BL21DE3pET42a-comD,E.coli BL21DE3pET42a-comE and E.coli BL21DE3pET32a-comC were able to efficiently express the target recombinant proteins and the outputs of rComD,rComE and rComC were 28%,25% and 35% of the total bacterial proteins,respectively.The double immunodiffusion titers of rabbit antisera against rComD,rComE or rComC were 1∶4,1∶4 and 1∶8,respectively.After the ComD and/or ComC were blocked by the antisera,the cefotaxime-sensitive S.pneumoniae strains became to resistant to antibiotics but there were no changes for cefotaxime-resistant strains and resistance to penicillin for all tested strains.Conclusion: The prokaryotic expression systems of S.pneumoniae comD/come/comC genes have been successfully constructed,and the study also indicates that both the ComD and ComC are involved in the drug resistance of S.pneumoniae to cefotaxime.更多还原