中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2014年
11期
844-848
,共5页
孙颜红%张少妮%楼永良%严杰%孙爱华
孫顏紅%張少妮%樓永良%嚴傑%孫愛華
손안홍%장소니%루영량%엄걸%손애화
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
肺炎鏈毬菌%phpP基因%錶達產物%PP2C型燐痠酶%β-內酰胺類抗生素
폐염련구균%phpP기인%표체산물%PP2C형린산매%β-내선알류항생소
Streptococcus pneumoniae%phpP gene%Expression product%PP2C type phosphatase%β-lactams antibiotics
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。
目的：構建肺炎鏈毬菌StkP／PhpP信號偶聯中燐痠酶編碼基因phpP原覈錶達繫統，瞭解錶達產物rPhpP燐痠酶活性及類型。方法採用PCR擴增肺炎鏈毬菌ATCC6306株phpP基因併測序。採用常規基因工程技術構建phpP基因原覈錶達繫統。採用SDS-PAGE及凝膠圖像分析繫統檢查rPhpP錶達情況及其可溶性，Ni-NTA親和層析法提純rPhpP。實時熒光定量RT-PCR檢測亞緻死量青黴素和頭孢噻肟藥物作用後phpP基因轉錄水平變化。採用專業生物信息學軟件分析phpP基因序列中燐痠酶功能結構域及其類型。採用絲／囌氨痠燐痠酶檢測試劑盒檢測rPhpP水解PP2C型燐痠酶活性併測定其酶動力學參數。結果剋隆的phpP基因序列與GenBank報道序列完全相同。所構建的phpP基因原覈錶達繫統錶達可溶性rPhpP。亞緻死量青黴素和頭孢噻肟藥物作用後phpP-mRNA水平升高。 phpP基因序列中含有PP2Cc燐痠酶結構功能域。提純的rPhpP能水解PP2C型燐痠酶底物燐痠肽[RRA（pT）VA]且活性隨rPhpP濃度增加而升高，其Km 和Kcat值分彆為277．35μmol／L和0．71 S－1。結論肺炎鏈毬菌phpP基因錶達產物為PP2C型燐痠酶，亞緻死量青黴素和頭孢噻肟可誘導phpP基因錶達上調。
목적：구건폐염련구균StkP／PhpP신호우련중린산매편마기인phpP원핵표체계통，료해표체산물rPhpP린산매활성급류형。방법채용PCR확증폐염련구균ATCC6306주phpP기인병측서。채용상규기인공정기술구건phpP기인원핵표체계통。채용SDS-PAGE급응효도상분석계통검사rPhpP표체정황급기가용성，Ni-NTA친화층석법제순rPhpP。실시형광정량RT-PCR검측아치사량청매소화두포새우약물작용후phpP기인전록수평변화。채용전업생물신식학연건분석phpP기인서렬중린산매공능결구역급기류형。채용사／소안산린산매검측시제합검측rPhpP수해PP2C형린산매활성병측정기매동역학삼수。결과극륭적phpP기인서렬여GenBank보도서렬완전상동。소구건적phpP기인원핵표체계통표체가용성rPhpP。아치사량청매소화두포새우약물작용후phpP-mRNA수평승고。 phpP기인서렬중함유PP2Cc린산매결구공능역。제순적rPhpP능수해PP2C형린산매저물린산태[RRA（pT）VA]차활성수rPhpP농도증가이승고，기Km 화Kcat치분별위277．35μmol／L화0．71 S－1。결론폐염련구균phpP기인표체산물위PP2C형린산매，아치사량청매소화두포새우가유도phpP기인표체상조。
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.
