牛布氏杆菌病和分枝杆菌病二联表位疫苗的构建及其免疫效果评价  

Construction and immunological evaluation of bovine brucellosis and mycobacteriosis bivalent epitope vaccine

作　　者：卢宝平 扈立伟 任君 杜荣起 顾天越 包利霞 刘青 朱凡杰 张东超[1,2] 金天明 LU Baoping;HU Liwei;REN Jun;DU Rongqi;GU Tianyue;BAO Lixia;LIU Qing;ZHU Fanjie;ZHANG Dongchao;JIN Tianming(Tianjin Key Laboratory of Agricultural Animal Breeding and Health Breeding,Tianjin Agricultural University,Tianjin 300392,China;College of Animal Science and Veterinary Medicine,Tianjin Agricultural University/Tianjin Engineering Technology Center of Livestock Pathogen Detection and Genetic Engineering Vaccine,Tianjin 300392,China;Tianjin Academy of Agricultural Sciences/Tianjin Key Laboratory of Molecular Breeding and Biotechnology of Livestock and Poultry,Tianjin 300192,China)

机构地区：[1]天津农学院/天津市农业动物繁育与健康养殖重点实验室,天津300392 [2]天津农学院动物科学与动物医学学院/天津市畜禽病原检测与基因工程疫苗工程技术中心,天津300392 [3]天津市农业科学院/天津市畜禽分子育种与生物技术重点实验室,天津300192

出　　处：《畜牧与兽医》2024年第2期69-75,共7页Animal Husbandry & Veterinary Medicine

基　　金：天津市自然科学基金重点项目(12JCZDJC22100);国家自然科学基金青年基金项目(32202760);天津市农委农业科技示范推广项目(批准文号:201202110);天津市兽医生物技术优秀科研创新团队项目(TD12-5019);天津市高等学校科技发展基金项目(20060720);天津市农业动物繁育与健康养殖重点实验室开放基金课题资助项目(2020zdkf02)。

摘　　要：为研发一种更安全、有效的防控牛布氏杆菌病和分枝杆菌病的疫苗,运用在线软件ABCPerd和IEBD对牛布氏杆菌外膜蛋白(Omp25)和牛分枝杆菌抗原85A(Ag85A)的B、T细胞表位进行预测分析,设计新的表位基因肽段NewⅠ和NewⅡ,并通过SOPMA和VaxiJen在线软件对NewⅠ和NewⅡ的二级结构与抗原性进行分析。将NewⅠ、Omp25、NewⅡ和Ag85A基因序列依次用自剪切肽T2A、P2A、E2A进行连接,获得串联基因并命名为COE,将COE基因构建至真核表达载体GV658,获得重组质粒GV658-COE;将上述重组质粒转染至HEK293细胞,利用细胞计数(CCK-8)试验评估GV658-COE对细胞的安全性,通过免疫印迹分析目的蛋白的表达情况;将重组质粒以400μg/只的剂量免疫大鼠,检测在第3次免疫后的血清抗体及大鼠脾淋巴细胞中的T细胞亚群占比情况。结果显示:NewⅠ和NewⅡ序列二级结构较稳定,抗原性良好;重组质粒经PCR扩增获得3231 bp大小的目的条带,与预计相符;重组质粒对HEK293细胞无毒性作用;重组质粒在HEK393细胞中可表达出目的蛋白,分别在22.3、17.3、23.9和35.6 kDa处有目的蛋白NewⅠ、Omp25、NewⅡ和Ag85A的表达。三免后,重组质粒免疫大鼠产生的IgG水平均高于空载质粒组和PBS组;与PBS组相比,重组质粒免疫大鼠脾脏中CD4^(+)和CD8^(+)T淋巴细胞百分比均升高。研究表明,重组质粒GV658-COE能有效激发大鼠的体液免疫和细胞免疫反应,可成为预防牛布氏杆菌病和分枝杆菌病的候选疫苗。In order to develop a safer and more effective vaccine against bovine brucellosis and mycobacteriosis,the epitopes of B and T cells of Brucella outer membrane protein Omp25 and Mycobacterium bovis antigen 85A(Ag85A)were predicted and analyzed using the online software ABCPerd and IEBD,and New epitope gene peptide segments NewⅠand NewⅡwere designed.The secondary structure and antigenicity of NewⅠand NewⅡwere analyzed by the SOPMA and VaxiJen online software.The gene sequences of NewⅠ,Omp25,NewⅡand Ag85A were connected with self-shear peptides T2A,P2A and E2A successively to obtain tandem genes which were named COE.The COE genes were constructed into the eukaryotic expression vector GV658,and the recombinant plasmid GV658-COE was obtained.The recombinant plasmid was transfected into HEK293 cells,the safety of GV658-COE was evaluated by CCK-8 assay,and the expression of the target protein was analyzed by Western blot.Finally,rats were immunized with the recombinant plasmid at 400μg/piece,and the serum antibodies and the proportion of T cell subsets in the spleen lymphocytes of the rats were detected after the third immunization.The results showed that the secondary structures of NewⅠand NewⅡsequences were stable and had good antigenicity.The target band size of 3231 bp was obtained by PCR amplification,which was consistent with that of the prediction.The recombinant plasmid had no toxic effect on HEK293 cells.The recombinant plasmid was able to express target proteins in HEK393 cells;and target proteins NewⅠ,Omp25,NewⅡand Ag85A are expressed at 22.3,17.3,23.9 and 35.6 kDa,respectively.After three rounds of immunization,the IgG levels of brucellosis and mycobacteriosis produced by the recombinant plasmid were higher than those in no-loaded plasmid group and the PBS group.Compared with the PBS group,the percentages of CD4^(+)and CD8^(+)T lymphocytes in spleen of the recombinant plasmid immunized rats were increased.The above results indicated that recombinant plasmid GV658-COE effectively stimulat

关 键 词：牛布氏杆菌病 牛分枝杆菌病 表位疫苗 Omp25 AG85A 

分 类 号：S852[农业科学—基础兽医学]