乙型脑炎病毒结构蛋白PrM单克隆抗体的制备和分析  

Development and analysis of monoclonal antibodies against PrM protein of Japanese encephalitis virus

作　　者：陈梦莉 谢丰玉 张彤 郑家杨 魏建超[2] 李宗杰 李蓓蓓[2] 邱亚峰[2] 马志永[2] 邵东华[2] 刘珂[2] 冯秀丽[1] CHEN Mengli;XIE Fengyu;ZHANG Tong;ZHENG Jiayang;WEI Jianchao;LI Zongjie;LI Beibei;QIU Yafeng;MA Zhiyong;SHAO Donghua;LIU Ke;FENG Xiuli(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《畜牧与兽医》2023年第6期76-81,共6页Animal Husbandry & Veterinary Medicine

基　　金：上海市自然科学基金面上项目(21ZR1477100)。

摘　　要：为了制备乙型脑炎病毒(Japanese encephalitis virus,JEV)结构蛋白PrM单克隆抗体,首先根据NCBI参考序列(GQ918133.2)设计PrM引物扩增目的基因,将目的基因构建至pColdⅠ原核表达质粒上;然后鉴定阳性克隆质粒并转化至BL21(DE3)感受态细胞,经诱导表达蛋白纯化后。免疫6周龄BALB/c小鼠,进行杂交瘤细胞融合,并对融合细胞进行克隆筛选;最后将筛选阳性的杂交瘤细胞制备腹水获取单克隆抗体,采用间接ELISA检测抗体效价,Western blot和间接免疫荧光检测抗体特异性和病毒感染过程。结果显示:成功构建了pCold I-PrM原核表达载体,PrM蛋白在1 mmol/L IPTG 16℃过夜诱导培养时表达量较高,亚克隆后经腹水制备获得2株针对PrM区域的单克隆抗体,Western blot和间接免疫荧光检测2株单抗能够特异性识别JEV的PrM区蛋白,获得的单抗能够鉴定JEV在细胞内的感染过程。本研究为进一步研究JEV鉴别诊断技术和生物学功能提供基础。In order to obtain Japanese encephalitis virus(JEV)PrM monoclonal antibodies,primers were designed according to the NCBI reference sequence to amplify its target sequence,and the target gene was constructed on the pColdI plasmid.The positive recombinant plasmid was transformed into BL21(DE3)competent cells;and then,BL21 was treated by adding IPTG for protein expression.The target protein was purified by nickel ion affinity chromatography.The purified proteins were used for mice immunity.Next,cell fusion,screening,and ascites preparation were performed to obtain monoclonal antibodies.The titer of the antibodies was measured by indirect ELISA,and the specificity of the antibodies was detected by Western blot and indirect immunofluorescence.The results were that a pCold I-PrM prokaryotic expression vector was successfully constructed.The PrM protein showed the highest expression at 1 mmol/L IPTG at 16℃overnight.Two monoclonal antibodies against the PrM regions were obtained by ascites preparation after sub-cloning,and the two monoclonal antibodies specifically recognized the PrM region proteins of JEV by Western blot and indirect immunofluorescence.The monoclonal antibodies were able to validate the intracellular infection process of JEV.In this study,the monoclonal antibodies against PrM protein of JEV provided a potential for further research on and detection of the Japanese encephalitis virus.

关 键 词：日本乙型脑炎病毒 PrM/M 单克隆抗体 

分 类 号：S852.6[农业科学—基础兽医学]