奶牛源无乳链球菌enolase重组蛋白制备及其免疫保护性研究  

Preparation and immunoprotective research of the recombinant protein enolase of Streptococcus agalactiae from dairy cows

作　　者：曾严 张保海 芦彪 韩光丽 张洁如 姚学萍[1] 王印[1,2] 杨泽晓[1] 罗燕[1] 曹随忠[1] ZENG Yan;ZHANG Bao-hai;LU Biao;HAN Guang-li;ZHANG Jie-ru;YAO Xue-ping;WANG Yin;YANG Ze-xiao;LUO Yan;CAO Sui-zhong(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Animal Disease and Human Health of Sichuan Province,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]动物疫病与人类健康四川省重点实验室,四川成都611130

出　　处：《中国兽医科学》2023年第7期902-910,共9页Chinese Veterinary Science

基　　金：四川农业大学学科建设双支计划项目(03571537);四川省科技厅科技支撑项目(2019YJ0650)。

摘　　要：奶牛乳房炎是奶牛养殖场常见疾病,为了有效预防奶牛乳房炎,降低药物残留,提高奶牛养殖的生产效益,为牛源无乳链球菌亚单位疫苗的制备提供一定的依据,本研究选取无乳链球菌免疫原性较好的enolase蛋白为靶标,克隆enolase基因、构建重组表达质粒。经过诱导、表达和纯化获得重组enolase蛋白。用纯化的蛋白加以佐剂对小鼠进行二次免疫,测定免疫后小鼠的血清抗体水平,评价enolase蛋白亚单位疫苗的免疫效果和安全性。最终,成功从无乳链球菌分离株MN686586中扩增出enolase基因,构建出重组表达质粒pET-32a-enolase,经IPTG体外诱导表达,获得分子质量大小为67 ku的重组蛋白。用enolase亚单位疫苗免疫小鼠,间接ELISA检测小鼠血清抗体水平显示,二免后第14天小鼠血清抗体水平效价达到1∶25 600,并且相对免疫保护率(RPS)为100%。结果表明,enolase亚单位疫苗能够刺激小鼠产生一定的免疫应答和产生特异性抗体,已初步制备出针对牛源无乳链球菌的亚单位疫苗。Cow mastitis is a common disease in dairy farms,which seriously damages the economic benefits of dairy farming.Vaccine is an effective way to prevent this disease,reduce drug residues and improve production efficiency.In this study,the enolase protein,which has the good immunogenicity of Streptococcus agalactiae,was selected as the target.We construct the recombinant expression plasmid pET-32a-enolase by cloning the enolase gene,and the recombinant protein was obtained after the induc-tion of IPTG in vitro,followed by the purification by NI-NTA column.The mice were immunized with puri-fied enolase recombinant protein,and the immunity was enhanced again on the 14th day after first immu-nization.Serum antibody levels were detected by indirect ELISA to evaluate the immune effect of enolase protein subunit vaccine on mice,and the immunoprotection efficiency was determined and expressed by relative percent survival(RPS).At last,the molecular weight of 67 ku recombinant protein was obtained.The titer of serum antibody levels at the 14th day after the second immunization reached 1∶25600.The RPS of the subunit vaccine was up to 100%.All data from this study suggested that a subunit vaccine against S.agalactiae of bovine has been initially prepared.

关 键 词：奶牛乳房炎 无乳链球菌 enolase蛋白 亚单位疫苗 

分 类 号：S852.659.6[农业科学—基础兽医学]