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  

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

作　　者：李丹丹[1] 杨春亮[2] 

机构地区：[1]西北农林科技大学动物医学院预防兽医学系免疫学教研室,杨凌712100 [2]哈尔滨医科大学基础医学学院,哈尔滨150081

出　　处：《中国生物工程杂志》2008年第11期14-19,共6页China Biotechnology

摘　　要：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CH86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HEVswDQ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 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 1：1.60×10^3-1：3.20×10^3 , respectively , and the titers in the ascites of the mice were 1：1.28×10^6-1：2.56×10^6, 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 poiyclonal antiserum against HEV. The result shows that McAb against HEV ORF2-V1 protein, its speciality is high. Six monoclonal antibody belonged to/gGl, The titers of the six McAbs in the supernatant culture supernatant were 1：1.60×10^3-1：3.20×10^3, and the titers in the ascites of the mice were 1：1.28×10^6-1：2.56×10^6. 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.

关 键 词：猪戊型肝炎病毒 ORF2-V1蛋白 单克隆抗体 识别位点 

分 类 号：Q78[生物学—分子生物学] S852.43[农业科学—基础兽医学]