肺炎链球菌phpP基因重组表达及其产物PP2C型磷酸酶活性的研究  

Prokaryotic expression of Streptococcus pneumoniae phpP gene and evaluation of PP2C type phosphatase activity of the expressed recombinant protein

作　　者：孙颜红[1] 张少妮[1] 楼永良[1] 严杰[2] 孙爱华[3] 

机构地区：[1]温州医科大学检验医学院和生命科学学院,325035 [2]浙江大学医学院病原生物学系 [3]浙江医学高等专科学校基础医学部,杭州310053

出　　处：《中华微生物学和免疫学杂志》2014年第11期844-848,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（81271893）;浙江省自然科学基金（LYl2H19002）;浙江省卫生厅科研项目（2011KYA005）

摘　　要：目的：构建肺炎链球菌StkP／PhpP信号偶联中磷酸酶编码基因phpP原核表达系统，了解表达产物rPhpP磷酸酶活性及类型。方法采用PCR扩增肺炎链球菌ATCC6306株phpP基因并测序。采用常规基因工程技术构建phpP基因原核表达系统。采用SDS-PAGE及凝胶图像分析系统检查rPhpP表达情况及其可溶性，Ni-NTA亲和层析法提纯rPhpP。实时荧光定量RT-PCR检测亚致死量青霉素和头孢噻肟药物作用后phpP基因转录水平变化。采用专业生物信息学软件分析phpP基因序列中磷酸酶功能结构域及其类型。采用丝／苏氨酸磷酸酶检测试剂盒检测rPhpP水解PP2C型磷酸酶活性并测定其酶动力学参数。结果克隆的phpP基因序列与GenBank报道序列完全相同。所构建的phpP基因原核表达系统表达可溶性rPhpP。亚致死量青霉素和头孢噻肟药物作用后phpP-mRNA水平升高。 phpP基因序列中含有PP2Cc磷酸酶结构功能域。提纯的rPhpP能水解PP2C型磷酸酶底物磷酸肽[RRA（pT）VA]且活性随rPhpP浓度增加而升高，其Km 和Kcat值分别为277．35μmol／L和0．71 S－1。结论肺炎链球菌phpP基因表达产物为PP2C型磷酸酶，亚致死量青霉素和头孢噻肟可诱导phpP基因表达上调。Objective To construct a prokaryotic expression system for expressing the phosphatase-encoding gene phpP in StkP/PhpP signaling couple in Streptococcus pneumonia （ S.pneumoniae） strains, and to further understand the phosphatase activity of the recombinant protein rPhpP.Methods The entire phpP gene of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR products were sequenced.A prokaryotic ex-pression system for expressing the phpP gene was constructed by the genetic engineering technique.The ex-pressed protein rPhpP and the solubility of rPhpP were assessed by SDS-PAGE and gel image analyzer.Ni-NTA affinity chromatography was performed to purify rPhpP.The changes of phpP gene transcription after the treat-ment with sublethal dosages of penicillin and cefotaxime were determined by real-time fluorescent quantitative RT-PCR.The functional domain in the sequence of the phpP gene and its type was analyzed by bioinformatic softwares.The activity of rPhpP in hydrolyzing the substrate of PP2C phosphotase was measured with Serine/Threonine Phosphotase Assay Kit.The enzyme kinetic parameters of rPhpP were calculated.Results The se-quence of the cloned phpP gene was identical with that reported in GenBank.The rPhpP in soluble form was ex-pressed in the constructed prokaryotic expression system.An increased expression of phpP gene at mRNA level was induced by sublethal dosage of penicillin or cefotaxime.The domain of PP2Cc type phosphatase was detec-ted in the sequence of phpP gene.The purified rPhpP protein could hydrolyze phosphopeptides [ RRA （ pT） VA], a substrate of PP2C type phosphatase, in a dose-dependent manner with Km and Kcat values of 277.35μmol/L and 0.71 S-1 ,respectively.Conclusion The protein encoded by phpP gene of S.pneumoniae was a PP2C type phosphatase.The expression of phpP gene could be enhanced by sublethal dosage of penicillin or ce-fotaxime.

关 键 词：肺炎链球菌 phpP基因 表达产物 PP2C型磷酸酶 Β-内酰胺类抗生素 

分 类 号：R378.12[医药卫生—病原生物学]