DNA免疫制备猪传染性胃肠炎病毒S蛋白单克隆抗体

Preparation of monoclonal antibodies against recombinant S protein of TGEV with DNA immunization

利用含有猪传染性胃肠炎病毒S基因主要抗原位点的重组质粒PCI-Sa免疫BALB/c小鼠,通过细胞融合技术,间接ELISA方法进行筛选和有限稀释法3次克隆,获得1株稳定分泌抗TGEV重组S蛋白单克隆抗体的杂交瘤细胞株,命名为E6D9A12,其染色体平均计数为88±10对,间接ELISA检测制备腹水抗体效价为1:6×105,分泌抗体亚类为IgG2a型。杂交瘤细胞上清与猪流行性腹泻病毒(PEDV)、猪轮状病毒(PRV)、猪伪狂犬病病毒(PrV)均不发生交叉反应;与TGEV感染细胞经间接免疫荧光检测均呈黄绿色荧光;与TGEV经Dot-ELISA检测呈特异性反应。

Hybridomas secreting monoclonal （MAB） to S protein of transmissible gastroenteritis virus （TGEV） were prepared by fusion of SP2/0 myeloma cells and splenic lymphocytes of BALB/c mice immunized with recombinant plasmid named PCI-Sa. The hybridoma supernatants were selected for positive cells of selecting antibody. A hybridoma cell line（designated E6D9A12） producing antibodies against TGEV was obtained by limiting dilution and three serials of clones. The average number of chromosomes was 88±10 pairs. The antibody titer of ascites was 1：6×10^5 and no cross-reaction between the McAbs with PEDV, PRV and PrV was observed. The result of IFA indicated that cells infected by TGEV showed strongly yellow and green fluorescence after reacting with McAbs. The antibody specifically reacted with purified TGEV in Dot-ELISA.