猪带绦虫丝氨酸蛋白酶抑制剂4848的鉴定及表达

发布时间：2019-04-04 08:56

【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。
[Abstract]:In order to identify and express serine protease inhibitor (TsSerpin), of Taenia solium, primers were designed to extract total RNA, from cysticercus cellulosae and TsSerpin4848 gene was amplified by RT-PCR. The purified PCR products were ligated into p-MD19-T vector and transformed into E. coli DH5 competent cells. The positive clones were screened and sequenced, and their sequence and structural characteristics were analyzed by bioinformatics method. The recombinant expression vector p ET-30a ()-4848 was constructed, identified by restriction endonuclease digestion and sequencing, and then transformed into E. coli BL21 (DE3) for induced expression. The target protein was purified and analyzed by SDSPAGE and Western-blot. The results show that the open reading frame of TsSerpin4848 consists of 1 065 nucleotides and encodes 355amino acid residues. Its theoretical molecular weight is about 39 ku.TsSerpin4848 containing the conserved sequences DEEGAE and FIVDHPFLFFI, of serpin family and the specific reaction center domain of serpin. There are 8 helices and 3 尾 folds in the tertiary structure. The recombinant TsSerpin4848 protein was mainly in the form of inclusion body in E. coli and could be recognized by the positive serum of cysticercus cellulosae. It indicated that the TsSerpin4848 protein had good reactivity and could be used as a candidate antigen for the diagnosis of cysticercosis.
【作者单位】：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;
【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)
【分类号】：S852.734

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