牛病毒性腹泻病毒重组抗原对小鼠的免疫效果评价  

Evaluation of immune effect of BVDV recombinant antigens in mice

作　　者：李玉豪 钱天皓 刘浩 李崇池 郑婷 宋雯妍 齐亚银[1] 盛金良[1] LI Yu-hao;QIAN Tian-hao;LIU Hao;LI Chong-chi;ZHENG Ting;SONG Wen-yan;QI Ya-yin;SHENG Jin-liang(College of Animal Science and Technology,Shihezi University,Shihezi 832001,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832001

出　　处：《中国兽医科学》2022年第11期1437-1443,共7页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31260610,31760737);新疆生产建设兵团科技攻关项目“犊牛健康养殖关键技术继承与示范”(2018AB041);新疆生产建设兵团科技发展专项资金“牛病毒性腹泻黏膜病(BVDV)快速诊断试剂盒的研发与应用”(2017BA044);十二师重大科技项目任务书“南北疆部分规模化奶牛场BVDV和IBR的调查及综合防控技术的推广应用”(SR2019002)。

摘　　要：为成功研制BVDV重组抗原蛋白(NS5A、E0、E2、mE2T)亚单位疫苗并对比分析其免疫效价,本研究对BVDV的4种不同功能蛋白进行原核表达后,与弗氏佐剂乳化进而研制成亚单位疫苗。然后,选择105只BALB/c小鼠为试验动物,将其随机分成7组进行3次免疫,以便从细胞免疫和体液免疫方面评估其免疫效果。在第3次免疫后第14天对小鼠进行攻毒,通过qRT-PCR方法测定小鼠体内BVDV病毒的拷贝量。结果,成功研制出BVDV重组亚单位疫苗。3次免疫后,与免疫前相比,各组小鼠白细胞数量显著上升(P<0.05),其中联合疫苗组攻毒前、后白细胞数量最高,攻毒前为6.48±0.19,攻毒后为6.08±0.82。在3次免疫之后,联合疫苗组的抗体水平最高,在倍比稀释后为0.573±0.055,灭活疫苗为0.552±0.027;亚单位疫苗组中mE2T组的抗体水平最高,为0.535±0.028。MTT法检测小鼠脾淋巴细胞增殖时发现,联合疫苗组的SI值最高,为1.963。抗体中和试验证明,各个疫苗组的小鼠血清对病毒均有一定的中和能力,其中联合疫苗组的中和能力最强,达到了1∶18.2。qRT-PCR分析小鼠体内病毒的拷贝量,发现联合疫苗组小鼠体内病毒拷贝量最低,为1×10^(2.3)/mL。上述研究结果为BVDV的预防和控制提供了基础,也为新型疫苗的研制提供了依据。In order to develop bovine viral diarrhea virus(BVDV)recombinant antigen protein(NS5A,E0,E2,m E2T)subunit vaccine and to analyze its immunotiter,in this study,four different functional proteins of BVDV were expressed prokaryotically and emulsified with Freund’s adjuvant to prepare subunit vaccines.Then,105 BALB/c mice were randomly divided into 7 groups for three times of immunization,and the effect was evaluated from the aspects of cellular and humoral immunity.On the 14th day after the third immunization,the mice were challenged with the virus,and the copy quantity of BVDV in mice was determined by q RT-PCR.In result,BVDV recombinant subunit vaccines were successfully developed.After3 times of immunization,the number of white blood cells in each group was significantly increased compared with that before immunization(P<0.05),and the number of white blood cells in the combined vaccine group was the highest before and after challenge(6.48±0.19 before and 6.08±0.82 after challenge).After 3immunizations,the highest antibody level was 0.573±0.055 in the combined vaccine group,0.552±0.027in the inactivated vaccine group,and 0.535±0.028 in the m E2T subunit vaccine group.The proliferation of splenic lymphocytes was detected by MTT assay.It was found that the combined vaccine group had the highest SI(1.963).The antibody neutralization test proved that the mouse serum of each vaccine group had certain neutralization ability to virus,among which the neutralization ability of the combined vaccine group was the strongest,reaching 1∶18.2.q RT-PCR analysis of virus copy volume in mice showed that the virus copy volume in the combined vaccine group was the lowest,which was 1×10^(2.3)/m L.The results provide a basis for the prevention and control of BVDV,and also provide a basis for new vaccines and methods.

关 键 词：牛病毒性腹泻病毒 灭活疫苗 亚单位疫苗 小鼠 

分 类 号：S852.659.6[农业科学—基础兽医学]