携带猪囊虫表位的嵌合蛋白疫苗的构建及其免疫学研究  

Construction and Immune Research of a New Chimeric Protein Vaccine Containing Epitopes of Taenia solium Cysticercus

作　　者：邓小昭[1] 周宗安[1] 刁振宇[1] 高健[1] 王元伦[1] 刘玉[1] 郑纪山[1] 

机构地区：[1]南京军区军事医学研究所,江苏南京210002

出　　处：《中国兽医学报》2006年第3期288-291,共4页Chinese Journal of Veterinary Science

基　　金：江苏省"十五"攻关课题(BG2000003)

摘　　要：用PCR法将猪囊虫抗原表位n1、n2插入乙肝病毒核心蛋白（HBc）序列的第78～79位之间，将猪囊虫抗原表位n3接在149位之后，再将该序列克隆入pET28a载体中，构建了重组表达质粒pET28a—△c-3n，在大肠杆菌中表达出融合蛋白。命名为PCCE。将PCCE纯化后免疫小鼠，以ELISA检测小鼠的体液免疫应答，Western blot检测抗体的特异性．Dot ELISA验证所选表位的保护性。另用绦虫卵攻击免疫小鼠，以观察疫苗的保护作用。测序结果表明，重组质粒构建成功；SDS—PAGE显示，融合蛋白表达正确，电镜证实有蛋白颗粒形成。对免疫小鼠应用ELISA检测到高滴度抗体；Western blot结果表明。免疫鼠体内诱导出了3种特异性抗体；Dot ELISA结果表明，所逸表位对宿主可能具有保护性。绦虫卵攻击免疫小鼠试验表明，疫苗PCCE的相对保护率为89％。结论：成功地表达和纯化了以HBc为载体的携带3个猪囊虫表位的融合蛋白（PCCE），以PCCE作为疫苗免疫小鼠，能诱导较强的特异性体液免疫反应，免疫小鼠对绦虫卵攻击具有较好的免疫保护作用。The truncated HBc vector containing three epitopes against T. solium cysticercosis was inserted into pET28a to construct a recombinant plasmid pET28a-Ac-3n. The chimeric protein was expressed and purified. Then it was administered to immunize mice to investigate the humoral immune response and the anti-egg rate. By PCR, the epitopes n1,n2 were inserted into the sequence of amino acids 78-79 of HBc and epitope n3 was linked to the C-terminus of HBc149. The PCR product was cloned into pET28a to construct a recombinant plasmid pET28a-△c-3n. The fused protein was expressed in E. coll. The purified protein was termed PCCE and was used to immunize mice. Specific antibody titer was tested by ELISA. The antibody specification was analyzed by Western blot. Dot ELISA was performed to vertify the protection of the three epitopes. Immunized mice were challenged with infectious cysticercus eggs and the relative protective rate was calculated. Results of DNA sequencing showed that the recombinant plasmid was successfully constructed. A fused protein with correct molecular mass was expressed and confirmed by SDS-PAGE. The formation of protein particle was verified by electron microscopy. High antibody titers were elicited in mice immunized with PCCE, and three different antibodies were induced, serum specific antibody recognized the native peptide localized mainly in cyst wall cells, and there was no specific antibody towards the three epitopes in sera of infected pig and humans. The relative protective rate of PCCE was 89%, which was obtained by the challenge test. Conclusion： A vaccine expressed as a modified HBc vector containing three protective epitopes against cysticercosis has been successfully expressed and purified. The strong humoral immune response and high protective rate of vaccine PCCE in mice indicated the potential value in preventing from T. solium cysticercosis.

关 键 词：猪囊虫 疫苗 HBCAG 保护性抗原表位 免疫反应 

分 类 号：S852.7[农业科学—基础兽医学] S858.28[农业科学—兽医学]