日本血吸虫蛋白磷酸酶PP2C-1的原核表达及活性分析  

Expression of protein phosphatase PP2C-1 from Schistosoma japonicum and analysis of its activity

作　　者：冯金荣[1] 周桥[1] 毛勇安 尹西腾 徐凌峰[1] 王里媛 段义农[1] 

机构地区：[1]南通大学医学院病原生物学系,江苏南通226001

出　　处：《中国病原生物学杂志》2015年第1期34-37,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31100112);江苏高校优势学科建设工程资助项目(No.PAPD);南通大学大学生创新训练计划项目(No.201410304049Z)

摘　　要：目的构建日本血吸虫PP2C-1蛋白磷酸酶表达载体,并进行体外表达和酶活性分析。方法从日本血吸虫基因组数据库中找到一个可能编码PP2C-1蛋白磷酸酶的基因Sj-PP2C-1,设计引物并通过PCR技术扩增得到该基因,将其成熟编码区与pET28a表达载体连接后转化大肠埃希菌并用IPTG诱导表达,对表达产物的磷酸酶活性进行分析。结果成功构建了Sj-PP2C-1蛋白磷酸酶表达载体,其编码区能够在大肠埃希菌中表达分子质量单位为40ku的目的蛋白;采用V2460系统进行检测,Sj-PP2C-1具有PP2C磷酸酶活性。结论 Sj-PP2C-1基因原核表达产物具有蛋白磷酸酶活性,为研究PP2C类蛋白磷酸酶的功能打下了基础。Objectives To construct a prokaryotic expression plasmid containing the type 2Cprotein phosphatase gene of Schistosoma japonicumand to analyze the type 2Cprotein phosphatase activity of the recombinant protein. Methods A potential PP2 Cgene named Sj-PP2C-1was obtained from a database containing the S.japonicumgenome.The gene fragment containing the mature ORF of Sj-PP2C-1 was amplified with PCR and then cloned into the expression plasmid pET28 a.The recombinant plasmid was confirmed to be correct using PCR and restriction enzyme analysis.The plasmid,designated pET28a-Sj-PP2C-1,was sequenced and transformed into Escherichia coli Rosetta（DE3）.Its expression was then induced with isopropyl-β-D-thiogalactopyranoside（IPTG）.The expressed protein products were analyzed using SDSPAGE and their protein phosphatase activity was analyzed. Results A prokaryotic expression plasmid containing the SjPP2C-1gene fromS.japonicum was successfully constructed.The exogenous Sj-PP2C-1gene was expressed in E.coli and generated a recombinant protein with a molecular weight of 40 ku.The phosphatase activity of the recombinant SjPP2C-1protein was detected with a V2460 system,and results indicated that Sj-PP2C-1has type 2Cphosphatase activity.Conclusion The recombinant Sj-PP2C-1protein had typical type 2Cprotein phosphatase activity,which means that it can be used for further study of the function of PP2 Cproteins in S.japonicum.

关 键 词：血吸虫 日本 PP2C蛋白磷酸酶 克隆 表达 活性分析 

分 类 号：R383.24[医药卫生—医学寄生虫学]