猪戊型肝炎病毒重组蛋白ORF2-V1单克隆抗体的制备及抗原表位差异分析

用纯化的猪戊型肝炎病毒DQ株重组蛋白ORF2-V1免疫BALB/c小鼠，取免疫小鼠脾细胞与SP2/0 骨髓瘤细胞进行融合，经3次克隆和间接ELISA 筛选，获得了αC11,αC12,γH1,γF8,BC4和CH8 6株稳定分泌单克隆抗体的杂交瘤细胞株。通过间接ELISA 测定，单抗效价为: 细胞培养上清1∶1.60×103～1∶3.20×103，腹水为1∶1.28×106～1∶2.56×106; 经ELISA法测定，6株单克隆抗体均与重组蛋白ORF2-V1反应， 而不与ORF2其它部分片段的蛋白反应; 将试验中制备的MAbs分别用HEV swDQ株感染的A549细胞涂片进行IFA检测，同时分别用猪的阳性、阴性血清和SP2/0细胞上清做对照，制备的6株MAbs对感染细胞均呈现阳性反应，说明其可以与天然的病毒发生反应，并且与阳性多克隆血清比较，以单克隆抗体为一抗，IFA反应表现出良好的特异性。单抗的亚类鉴定结果表明，所有MAbs均为IgG1型，而且所有单克隆抗体的轻链均为κ链。单克隆抗体抗原识别位点分析结果表明，6株单抗分别针对4个不同抗原位点，McAb-γH1,BC4,CH8识别的位点各不相同，其中McAb-γH1,BC4识别的位点有部分重叠，McAb αC11,αC12,γF8识别同一位点，位于γH1,BC4识别位点的重叠区内。

The Preparation of Monoclonal Antibody against ORF2-V1 Recombinant Proteins of Swine Hepatitis E Virus and Differential Recognition of Antigenic Epitopes

Abstract The BALB/c mice were immuned with purified recombinant fusion protein （ORF2-V1）from swine Hepatitis E virus. The myeloma cell line SP2/0 was fused with the spleen of the immuned BALB/c mice, and the positive clones which produced McAbs against ORF2-V1 were screened by ELISA. The titers of the six McAbs in the supernatant culture supernatant were 1∶1.60×103～1∶3.20×103, respectively , and the titers in the ascites of the mice were 1∶1.28×106～1∶2.56×106, respectively, detected by ELISA. And six McAbs only could react with ORF2-V1, and but not react with other recombinant fusion protein from ORF2. Among them , The A549 cell culture affected with HEV was carried out comparative studies, which is detection of pathogene with indirect immunofluorescence by using monoclonal antibody against HEV ORF2-V1 protein and polyclonal antiserum against HEV. The result shows that McAb against HEV ORF2-V1 protein, its speciality is high. Six monoclonal antibody belonged to IgG1, The titers of the six McAbs in the supernatant culture supernatant were 1∶1.60×103～1∶3.20×103, and the titers in the ascites of the mice were 1∶1.28×106～1∶2.56×106. The six strains of McAbs were specific for three different kinds of epitopes on HEV. Among them, McAb-γH1, McAb-BC4 and McAb CH8 direct to different epitopes, respectively．The epitope recognized by McAb-γH1 overlapped partly that of McAb-BC4．McAb-αC11 and McAb-αC12 were identical to McAb b-γF8．The epitope of which was supposed to be locateed in the overlapping region between epitope-γH1 and epitope-BC4．