牛轮状病毒多克隆抗体制备及特性分析  

Preparation and characterization of polyclonal antibody against bovine rotavirus

作　　者：孙心如 孙敏 周洪婷 李素芬 张雪寒[2] 周金柱 贡嘎 索朗斯珠 李彬[2] SUN Xinru;SUN Min;ZHOU Hongting;LI Sufen;ZHANG Xuehan;ZHOU Jinzhu;GONG Ga;SUOLANG Sizhu;LI Bin(College of Animal Science,Tibet Agriculture&Animal Husbandry University,Nyingchi 860000,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China)

机构地区：[1]西藏农牧学院动物科学学院,西藏林芝860000 [2]江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室,江苏南京210014

出　　处：《畜牧与兽医》2024年第7期72-77,共6页Animal Husbandry & Veterinary Medicine

基　　金：江苏省农业科技自主创新资金项目[CX(23)1029];财政部和农业农村部:国家现代农业产业技术体系(CARS-37)。

摘　　要：旨在优化G6P[1]型牛轮状病毒(bovine rotavirus, BRV)在MARC-145细胞中的培养条件,制备其兔源多克隆抗体,并对制备的多抗进行效价检测及鉴定。对G6P[1]型BRV毒株NCDV进行不同病毒接种量及收毒时间优化,确定病毒在MARC-145细胞中的最佳培养条件;以1×10^(7) TCID_(50) NCDV活毒作为免疫原免疫成年兔,制备兔源多克隆抗体;通过间接ELISA检测多抗效价,并通过间接免疫荧光试验(IFA)及中和试验鉴定其特异性。结果:NCDV毒株以感染比0.1、感染时间24 h为最佳培养条件,病毒滴度可至4.9×10^(6) TCID_(50)/mL;间接ELISA结果显示,多克隆抗体的效价为1∶51 200,表明NCDV具有良好的免疫原性;IFA及中和试验结果表明,NCDV多克隆抗体与G6P[1]型、G9P[1]型和G10P[11]型BRV毒株均能发生特异性反应,存在良好的交叉中和效应,表明制备的多抗具有良好的反应性。综上,本试验成功优化了NCDV毒株在MARC-145细胞中的增殖条件,并制备了高效价的兔源多克隆抗体,为后续BRV感染的血清学诊断及疫苗研发奠定了理论基础。The aim of this study was to optimize the culture conditions of G6P[1]type bovine rotavirus(BRV)in MARC-145 cells,to prepare BRV rabbit polyclonal antibody,and to evaluate the titer of the prepared polyclonal antibody.The optimal culture conditions of NCDV in MARC-145 cells were determined by optimizing the multiplicity of infection and the infection time.Then,rabbit polyclonal antibody was prepared by immunizing adult rabbits with 1×10^(7) TCID_(50) NCDV.Next,the titer of the antibody was detected by ELISA,and its specificity was determined by IFA and neutralization test.The results of cytopathic viral titer and the expression of the viral protein showed that the multiplicity of infection of 0.1 and infection time of 24 h were the best culture conditions for NCDV,and the virus titer could reach 4.9×10^(6) TCID_(50)/mL.The ELISA results showed that the titer of the polyclonal antibody was 1∶51200,which indicated that NCDV live virus possessed good immunogenicity.The results of IFA and neutralization tests showed that polyclonal antibodies of NCDV could react specifically with G6P[1],G9P[1]and G10P[11]BRV strains,and produced a good cross neutralization effect.This experiment successfully optimized the culture conditions for NCDV in MARC-145 cells,and prepared highly effective rabbit polyclonal antibodies,which laid a theoretical foundation for serological diagnosis and vaccine development of BRV.

关 键 词：牛轮状病毒 NCDV毒株 MARC-145细胞 多克隆抗体 

分 类 号：S852.653[农业科学—基础兽医学]