重组球菌和球虫多表位抗原的表达及特性研究  

Expression and characterization of recombinant multi-epitope antigens of cocci and coccidia

作　　者：符立发 梁鸽 黄家艳 唐智慧 赵仕杰 张焱荣 郑璨璘 索朗斯珠 曹随忠[3] 别明江 王保宁[1] FU Li-Fa;LIANG Ge;HUANG Jia-Yan;TANG Zhi-Hui;ZHAO Shi-Jie;ZHANG Yan-Rong;ZHENG Can-Lin;SUOLANG Si-Zhu;CAO Sui-Zhong;BIE Ming-Jiang;WANG Bao-Ning(West China School of Basic Medical Science and Forensic Medicine,Sichuan University,Chengdu 610041,China;College of Animal Science,Xizang Agricultural and Animal Husbandry University,Linzhi 860000,China;College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu 610041,China;Editorial Board of Journal of Sichuan University(Medical Science Edition),Chengdu 610041,China)

机构地区：[1]四川大学华西基础医学与法医学院,成都610041 [2]西藏农牧学院动物科学学院,林芝860000 [3]四川农业大学动物医学院,成都611130 [4]四川大学华西公共卫生学院/华西第四医院,成都610041 [5]四川大学学报(医学版)编辑部,成都610041

出　　处：《四川大学学报(自然科学版)》2025年第2期494-500,共7页Journal of Sichuan University(Natural Science Edition)

基　　金：西藏自治区科技厅重点研发计划项目(XZ202201ZY0009N);四川省科技厅项目(2023YFQ0051)。

摘　　要：本研究旨在设计针对牛链球菌和牛球虫的联合多表位疫苗.通过生物信息学筛选两种病原体的T/B细胞优势表位基因序列,串联拼接后引入霍乱毒素B亚单位(CTB)作为分子内佐剂,设计得到多表位重组抗原CARR(CTB-AMA1-RodA-RodA).经密码子优化后,将CARR基因克隆至pET28a(+)质粒,转化至大肠杆菌BL21(DE3),成功构建了稳定的表达工程菌.SDS-PAGE显示工程菌可高效表达约90 kDa目标蛋白,Western blot验证表达蛋白能与抗CTB及抗牛链球菌抗体特异性结合,且20代内工程菌质粒保有率超80%.纯化后的CARR蛋白免疫蛋鸡,7 wk后ELISA检测显示特异性IgY抗体效价达1∶12800.体外中和实验证实,该抗体显著抑制牛链球菌生长,说明其具备功能性中和活性.结果表明,CARR重组蛋白可有效快速激发宿主体液免疫应答,是牛链球菌和牛球虫联合疫苗的有效候选抗原靶点.This study aimed to develop a combined multi-epitope vaccine targeting both Streptococcus bovis and Bovine coccidia.Using bioinformatics approaches,dominant T-cell and B-cell epitope gene sequences from the two pathogens were identified and tandemly linked,followed by the incorporation of cholera toxin B subunit(CTB)as an internal molecular adjuvant to design the recombinant antigen CARR(CTB-AMA1-RodA-RodA).Following codon optimization,the CARR gene was cloned into the pET28a(+)vector and transformed into E.coli BL21(DE3),thereby establishing a stable expression strain.SDS-PAGE analysis confirmed the recombinant strain expressed a target protein(approximately 90 kDa),and Western blot confirmed its specific binding to both anti-CTB and anti-Streptococcus bovis antibodies,with a plasmid retention rate above 80%after 20 generations.The purified CARR protein was used to immunize laying hens,and ELISA results demonstrated that the specific IgY antibody titer reached 1:12,800 after 7 weeks.In vitro neutralization assays confirmed that the antibodies significantly inhibited the growth of S.bovis,indicating potent functional neutralizing activity.These results suggest that the CARR recombinant protein can effectively and rapidly elicit a robust humoral immune response in the host.This study provides a promising candidate antigen and technical foundation for the development of a combined vaccine against S.bovis and E.bovis infections.

关 键 词：牛链球菌 牛艾美尔球虫 重组多表位抗原 表达特性 中和抗体 

分 类 号：R392.1[医药卫生—免疫学]