作者：姚利晓,陶丽红,杨冠珍,吴祥甫,蔡幼民,林矫矫
作者单位：中国农业科学院上海兽医研究所,农业部动物寄生虫学重点开放实验室,上海动物生物技术研究中心,中国农业科学院上海兽医研究所,农业部动物寄生虫学重点开放实验室,中国科学院上海生命科学研究院上海生化细胞所,,农业部动物寄生虫学重点开放实验室
摘要：目的在昆虫细胞中表达获得可溶性的日本血吸虫SjPP重组蛋白。方法提取日本血吸虫成虫总RNA,通过RT-PCR扩增出SjPP基因的编码区序列,将其定向克隆到供体质粒pFastBac HT-B中,构建重组杆状病毒供体质粒pFastBac-SjPP,转化入大肠埃希菌DH10Bac进行转座,提取重组杆粒Bacmid-SjPP,用阳离子脂质体法转染粉纹夜蛾(TN5B1-4)细胞。利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析重组蛋白表达情况。结果构建了含日本血吸虫SjPP基因的重组杆粒Bacmid-SjPP,转染TN5B1-4细胞后,获得有感染力的重组杆状病毒,并在TN5B1-4细胞中成功表达SjPP蛋白。该重组蛋白可被抗SjPP蛋白的兔血清识别。结论成功构建SjPP基因重组杆状病毒,并在TN5B1-4细胞中表达,该蛋白具有良好的抗原性。
关键词：日本血吸虫,SjPP,Bac-to-Bac杆状病毒表达系统
Title: Enzymatic activities of the protein expressed-prokaryoticly in Escherichia coli from SjPP gene of Schistosoma japonicum
Authors: YAO Li-xiao,WANG Xin-zhi,FU Zhi-qiang,TAO Li-hong,LIN Jiao-jiao
Abstract: Using phosphothreonine polypeptide K-R-pT-I-R-R as substrate,the activities of Ser/Thr protein phosphatase(rSjPP) of Schistosoma japonicum expressed in Escherichia coli were tested.The effects of temperature,pH value,divalent cations(Mn2+,Ni2+,Mg2+,and Ca2+) on the enzymatic activities of the rSjPP were analyzed using pNPP as substrate.The phosphothreonine polypeptide could been hydrolyzed by the rSjPP,indicating that the rSjPP possessed the characteristics of Ser/Thr protein phosphatase.Using pNPP as substrate,rSjPP had the strongest catalytic activities under the condition of pH8.0 and 60 ℃.The activities of rSjPP could be enhanced by adding Ni2+ from 1 mmol/L to 4 mmol/L but not by Mn2+,Mg2+,and Ca2+
Key words: Schistosoma japonicum； Ser/Thr protein phosphatase； enzymatic activity； impact factor；
文献注录：姚利晓,陶丽红,杨冠珍,吴祥甫,蔡幼民,林矫矫. 日本血吸虫SjPP基因重组杆状病毒的构建和在昆虫细胞中的表达 [J]. 中国寄生虫学与寄生虫病杂志. 2008, 02: 124-127.
基金: 国家“863”计划(No.2006AA10A207);国家科技支撑计划(No.2006-BAD06A09)~~
报/刊名：《中国寄生虫学与寄生虫病杂志》，发表于2008 / 第 02 期。
文献类型： [J]
页码：第 124-127 页 / 共4 页