脑多头蚴TPx基因的克隆表达及免疫原性分析  

Cloning,Expression and Immunogenicity Analysis of TPx Gene of Coenurus Cerebralis

作　　者：李永光 曹艳桃 LI Yongguang;CAO Yantao(Dingxi Vocational and Technical College,Lintao 730500,China;College of Applied Technology,Gansu Agricultural University,Lintao 730500,China)

机构地区：[1]定西职业技术学院,临洮730500 [2]甘肃农业大学应用技术学院,临洮730500

出　　处：《中国草食动物科学》2024年第4期55-61,共7页China Herbivore Science

基　　金：甘肃农业大学科技创新基金(GAU-STS-2019-5)。

摘　　要：为了研究脑多头蚴(Coenurus cerebralis,Cc)硫氧还蛋白过氧化物酶(Thioredoxin peroxidase,TPx)的免疫原性,采集绵羊脑多头蚴原头蚴,提取总RNA;根据GenBank中亚洲牛带绦虫TaHc2-D11 mRNA序列设计特异性引物,RT-PCR扩增脑多头蚴TPx基因,构建重组质粒pMD18-T-CcTPx,鉴定后克隆至原核表达载体pGEX-4T-1中,构建的重组表达质粒pGEX-4T-1-CcTPx鉴定正确后转化至大肠杆菌BL21(DE3)中诱导表达;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测脑多头蚴TPx重组蛋白,纯化脑多头蚴TPx重组蛋白后皮下多点注射免疫绵羊,ELISA测定其血清抗体。结果表明,脑多头蚴TPx基因序列长614 bp,含有1个591 bp的、完整的开放阅读框,脑多头蚴TPx重组蛋白分子质量为47 kD,主要以包涵体形式存在,免疫接种绵羊可刺激机体产生抗体,二免或三免后抗体水平逐步升高,抗体水平显著高于对照组同期免疫脑多头蚴原头蚴抗原的绵羊。说明脑多头蚴TPx重组蛋白具有较强的免疫原性,可作为脑多头蚴疫苗的候选抗原。To study the immunogenicity of Thioredoxin peroxidase(TPx)for Coenurus cerebralis(Cc),the protoscoleces of Coenurus cerebralis were collected in sheep brain,and total RNA was extracted.Specific primers were designed according to the Taenia asiatica TaHc2-D11 mRNA sequences in GenBank,the Cc TPx gene was amplified by RTPCR,and the recombinant plasmid pMD18-T-CcTPx was constructed,then cloned into the prokaryotic expression vector pGEX-4T-1.The recombinant expression plasmid pGEX-4T-1-CcTPx was correctly identified and transformed into E.coli BL21(DE3)for induction of expression,and the Cc TPx recombinant protein was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).The purified Cc TPx recombinant protein was immunized in sheep by subcutaneous multi-point injection,and the antibodies in serum were detected with ELISA.The result showed that the Cc TPx gene was 614 bp in length and comprised an open reading frame(ORF)of 591 bp.The molecular mass of Cc TPx recombinant protein was 47 kD and it existed mainly in the form of inclusion bodies.The recombinant protein could stimulate sheep to produce antibodies,and the antibody level gradually increased after the second or third immunization,and the antibody level was significantly higher than that of the control group in sheep,which were immunized with the protoscoleces of Coenurus cerebralis at the same time.In summary,the recombinant protein of Cc TPx has strong immunogenicity and can be used as a candidate antigen for the Coenurus cerebralis vaccine.

关 键 词：脑多头蚴 TPx 重组蛋白 免疫原性 疫苗 

分 类 号：S852.7[农业科学—基础兽医学]