猪带绦虫丝氨酸蛋白酶抑制剂4848的鉴定及表达  

Identification and expression of Taenia solium serine protease inhibitor 4848

作　　者：梁盼红 刘光学[1] 毛立[1] 张少华[1] 王莉杰 王立群 骆学农[1] 

机构地区：[1]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》2017年第12期1486-1491,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0501303);国家自然科学基金面上项目(31372433)

摘　　要：为鉴定并表达猪带绦虫丝氨酸蛋白酶抑制剂(TsSerpin),提取猪囊尾蚴总RNA,设计引物并通过RT-PCR技术扩增TsSerpin4848基因,将纯化的PCR产物连接到p MD19-T载体后转化大肠杆菌DH5ɑ感受态细胞,筛选阳性克隆并测序,并用生物信息学方法分析其序列和结构特征。构建p ET-30a(+)-4848重组表达载体,经酶切鉴定及测序验证后转入大肠杆菌BL21(DE3)中进行诱导表达,目的蛋白纯化后进行SDSPAGE和Western-blot分析。结果表明,TsSerpin4848的开放阅读框由1 065个核苷酸组成,编码355个氨基酸残基,其理论分子质量约为39 ku。TsSerpin4848含有serpin家族保守序列DEEGAE和FIVDHPFLFFI,并含有serpin特有的反应中心环结构域,其三级结构中有8个ɑ螺旋和3个β折叠;重组TsSerpin4848蛋白在大肠杆菌中主要以包涵体形式存在,可被猪囊尾蚴阳性血清识别,表明TsSerpin4848蛋白具有良好的反应原性,有望作为猪囊虫病诊断的候选抗原。To identify and express the serine protease inhibitor of Taenia solium（TsSerpin）,the total RNA was extracted from T. solium metacestodes. Specific primers were designed and used to amplify TsSerpin4848 by RT-PCR. PCR products were ligated into the pMD19-T vector. After being transformed into Escheriehia coli DH5a,the positive clones were identified and confirmed by sequencing. The sequence of TsSerpin4848 was analyzed with the bioinformatics software. The recombinant pET-30a（＋）-4848 plasmid was constructed and transformed into E. coli BL21（BE3）.The target protein was expressed,purified and ana- lyzed by SDS-PAGE and Western-blot. In result,TsSerpin4848 cDNA sequence contained an open reading frame of 1 065 nucleotides,and the deduced protein consisted of 355amino acids with the theoretical protein molecular weight of 39 ku. TsSerpin4848 had a serpin conserved structure domain with the DEEGAE and FIVDHPFLFFI,and a specific reactive centre loop （RCL） motif. Analysis of tertiary structure of TsSerpin- 4848 revealed that it had eight a-helices and three g-sheets. The expressed products of TsSerpin4848, existed in inclusion bodies,could be recognized by the positive serum from the pig infected with solium by immunoblotting,indicating that the protein TsSerpin4848 has a good immunogenicity and it is hopeful to be a diagnostic antigen for cysticercosis.

关 键 词：猪带绦虫 丝氨酸蛋白酶抑制剂 鉴定 表达 候选抗原 

分 类 号：S852.734[农业科学—基础兽医学]