旋毛虫TsP53抗原基因的原核表达及抗原性分析  

Prokaryotic expression and antigenicity analysis of TsP53 gene of Trichinella spiralis

作　　者：王鸿盛[1,2] 李文卉[2] 盖文燕[2] 姚菊霞[2] 曲自刚[2] 王艳华[2] 张德林[2] 薛慧文[1] 付宝权[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点开放实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2010年第4期373-377,共5页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2007BAD40B03);中央级公益性科研院所基本科研业务费专项(0032007012);甘肃省科技重大专项(0702NKDA039);教育部留学回国人员科研启动基金资助项目

摘　　要：应用RT-PCR方法从旋毛虫肌幼虫总RNA中获得TsP53基因片段,限制性酶切后连接到表达质粒pET30a中,转化大肠杆菌DH5α。筛选阳性克隆,经限制性酶切、PCR分析及测序鉴定后,将重组表达质粒pET30a-TsP53转化大肠杆菌BL21(DE3),以IPTG诱导表达。SDS-PAGE分析表达产物,对表达产物纯化后通过间接ELISA鉴定重组蛋白的抗原性。结果显示,成功构建了重组表达质粒pET30a-TsP53,测序结果表明插入片段为1 176 bp,编码391个氨基酸残基,与旋毛虫P53抗原基因序列(U25127)有99%的同源性,开放阅读框正确。含有pET30a-TsP53重组表达质粒的大肠杆菌BL21(DE3)诱导后得到了高效表达,SDS-PAGE显示表达产物分子质量约为50 ku,主要以包涵体形式存在。间接ELISA结果表明,重组抗原可以被旋毛虫感染不同时期的猪血清特异性识别,有望用于旋毛虫病的免疫诊断。A TsP53 gene fragment was amplified by RT-PCR from the total RNA of Trichinella spiralis muscle larvae and cloned into the expression vector pET30a.It was identified by PCR,restriction enzyme digestion and sequencing,after transformation into Escherichia coli DH5α.The pET30a-TsP53 plasmid was transformed into E.coli BL21（DE3） and induced by IPTG for expression.The expressed product was analyzed by SDS-PAGE and the antigenicity of the purified recombinant protein was identified by indirect ELISA with T.spiralis-infected swine serum.It was demonstrated that after restriction enzyme digestion,PCR amplification and sequencing analysis,the constructed pET30a-TsP53 contained an insert of 1176bp encoding 391 amino acids,which was 99% homologous to T.spiralis P53 gene in GenBank.The recombinant protein was highly expressed in the form of inclusion bodies with the molecular weight of about 50ku.

关 键 词：旋毛虫 TsP53 原核表达 抗原性 

分 类 号：S852.731[农业科学—基础兽医学]