猪瘟与口蹄疫二联疫苗的制备及其免疫效果评价  

Development and immunogenicity evaluation of a bivalent vaccine for classical swine fever and foot-and-mouth disease

作　　者：高洁 袁红[1,2] 焦云娟 李坤 李平花[1,2] 杨宇轩 黄书伦[1,2] 郭海洋 邱酉飞 包慧芳 孙普 李冬 刘在新[1,2] 卢曾军 白兴文[1,2,3,4] GAO Jie;YUAN Hong;JIAO Yun-juan;LI Kun;LI Ping-hua;YANG Yu-xuan;HUANG Shu-lun;GUO Hai-yang;QIU You-fei;BAO Hui-fang;SUN Pu;LI Long;LIU Zai-xin;LU Zeng-jun;BAI Xing-wen(State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;WOAH(World Organization for Animal Health)/National Reference Laboratory for Foot and Mouth Disease,Lanzhou 730046,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,甘肃兰州730000 [2]WOAH/国家口蹄疫参考实验室,甘肃兰州730046 [3]甘肃省病原生物学基础学科研究中心,甘肃兰州730046 [4]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《中国预防兽医学报》2025年第2期172-178,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2021YFD1800300);甘肃省科技计划项目(23JRRA1515);甘肃省重大科技专项(21ZD3NA001、22ZD6NA001、22ZD6NA012);国家生猪技术创新中心项目(NCTIP-XD/C03)。

摘　　要：为研制猪瘟(CSF)与口蹄疫(FMD)二联疫苗并评价其免疫效果,本研究采用真核表达系统瞬时表达猪瘟病毒(CSFV)E2蛋白后经AKTA蛋白纯化仪和Hi Trap TALON纯化柱纯化,采用SDS-PAGE检测重组E2蛋白(r E2)的表达与纯化效果,采用western blot检测r E2与His-Tag单克隆抗体(MAb)的反应原性。结果显示,在约50 ku处出现目的蛋白条带,纯化后在约50 ku处出现单一条带。western blot结果显示在50 ku处出现特异性条带。表明,r E2获得了表达且纯化效果较好,反应原性较强。将纯化后的r E2与灭活的口蹄疫病毒(FMDV)O/Tibet/99株混合后经ISA 201佐剂乳化制备二联疫苗,将小鼠分为4组:CSFV E2+FMDV(20μg^(+)1μg)组、CSFV E2(20μg)组、FMDV(1μg)组及PBS阴性对照组。于第1 d和21 d分别将相应疫苗以肌肉注射方式免疫6周龄~8周龄的各组雌性BALB/c小鼠,首免后7 d、14 d、21 d、28 d、35 d、42 d采血,利用ELISA检测各组小鼠血清中的抗体水平;利用流式细胞术检测免疫后42 d各组小鼠血液中CD3^(+)/CD4^(+)T淋巴细胞、CD3^(+)/CD8^(+)T淋巴细胞和CD19^(+)B淋巴细胞的占比;采用ELISA检测免疫后42 d各组小鼠血清中IFN-γ的分泌水平。抗体检测结果显示,与PBS组相比,E2组、FMDV组和E2+FMDV组小鼠产生的针对两种病毒的抗体水平均显著和极显著升高(P<0.05、P<0.01、P<0.0001);E2+FMDV组与E2组和FMDV组小鼠血清中CSFV和FMDV的抗体水平均无显著差异(P>0.05)。流式细胞术结果显示,E2组、FMDV组和E2+FMDV组小鼠CD3^(+)/CD4^(+)T淋巴细胞、CD3^(+)/CD8^(+)T淋巴细胞及CD19^(+)B淋巴细胞的占比均显著和极显著高于PBS组(P<0.05、P<0.01、P<0.0001);E2+FMDV组与E2组小鼠上述指标均无显著差异;与FMDV组相比,E2+FMDV组小鼠CD3^(+)/CD4^(+)T淋巴细胞的占比极显著升高(P<0.01),而另外两个指标均无显著差异。ELISA检测结果显示,3个实验组小鼠血清中IFN-γ的分泌水平均显著和极显著高于PBS组(P<0.05、P<0.01),E2+FMDV组In order to develop a bivalent vaccine against classical swine fever(CSF)and foot and mouth disease(FMD)and evaluate its immunogenicity,the E2 protein of swine fever virus(CSFV)was expressed by eukaryotic expression system and purified using an AKTA purification system and a HiTrap TALON affinity column.The expression and purification efficiency of the recombinant E2 protein(rE2)were detected by SDS-PAGE.Additionally,the reactivity of rE2 with His-Tag monoclonal antibody was confirmed by western blot.The results showed a target protein band at approximately 50ku,and a single band at the same molecular weight was observed after purification.Western blot analysis revealed a specific band at 50ku,indicating correct expression of rE2.These findings demonstrated that rE2 was effectively expressed,purified,and had strong reactivity.The purified rE2 was mixed with inactivated foot-and-mouth disease virus(FMDV)strain O/Tibet/99,and the bivalent vaccine was prepared with ISA 201 adjuvant.The experiment was divided into 4 groups:CSFV E2+FMDV(20μg+1μg)group,CSFV E2(20μg)group,FMDV(1μg)group and PBS negative control group.Female BALB/c mice aged 6 weeks-8 weeks were immunized intramuscularly on day 1 and day 21.Blood samples were collected at 7 days,14 days,21 days,28 days,35 days and 42 days after the first dose immunization,and ELISA was used to detect the antibody levels of each group.The proportion of CD3+/CD4+T lymphocytes,CD3+/CD8+T lymphocytes and CD19+B lymphocytes were detected by flow cytometry at 42 days post-immunization.Additionally,the level of IFN-γsecretion levels in mice was detected by ELISA.The results showed that the antibody levels in E2 group,FMDV group and E2+FMDV group were significantly increased compared with PBS group(P<0.05,P<0.01,P<0.0001).Compared with E2 group and FMDV group,CSFV-and FMDV-specific antibody levels in E2+FMDV group were no significant difference(P>0.05).Flow cytometry results showed on day 42 post-immunization demonstrated that the proportion of CD3+/CD4+T lymphocytes,CD3+/C

关 键 词：猪瘟 E2蛋白 口蹄疫 二联疫苗 

分 类 号：S852.65[农业科学—基础兽医学]