猪传染性胃肠炎病毒S蛋白D位点单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibodies against S protein D site of transmissible gastroenteritis virus

通过大肠杆菌表达了猪传染性胃肠炎病毒(TGEV)S蛋白的D位点,并以D位点的六聚体作为抗原,免疫BALB/c小鼠,取免疫后小鼠脾细胞和骨髓瘤细胞SP2/0进行融合,经间接ELISA筛选和3次有限稀释法进行克隆。结果显示,得到3株能稳定分泌抗TGEV S蛋白D位点单克隆抗体的杂交瘤细胞株,分别命名为2C104、5E1012和4B101,其中2C104和5E1012分泌抗体类型是IgM亚类,4B101为IgG1亚类。2C104、5E1012、4B101三株杂交瘤细胞培养上清液的间接ELISA抗体效价分别为1∶128、1∶128、1∶64,腹水效价分别为1∶103、1∶104、1∶4×103。间接免疫荧光鉴定结果表明,3株细胞均能和TGEV发生特异性反应,且在连续传代和冻存后仍能稳定分泌抗体。本研究针对S蛋白D位点制备了单克隆抗体,为传染性胃肠炎的诊断和TGEV与宿主互作机制的研究奠定了基础。

Recombinant spike protein D site of transmissible gastroenteritis virus（TGEV） was ex- pressed in Escherichia coli BL21（DE3） and was used to immunize BALB/c mice. Cell fusion of spleen cell of the immunized mice with SP2/0 was performed according to a traditional method. Indirect ELISA was carried out to screen hybridoma cell lines by limiting dilution and 3 serials of clones,2C104,5E1012 and 4B101,were obtained respectively. The antibodies of 3 cell lines were respectively classed to IgM, IgM, IgG1 subgroups. The titers of 3 cell-cultured antibodies measured through indirect ELISA were to be 1 ＂ 128,1 ＂ 128,1 ： 64,and titers of antibodies produced in ascites were respectively 1 ： 103 ,1 ： 104 ,1 ： 4X 103. Antibodies were found to specifically bind to ST cell monolayers infected with TGEV with the confirmation of indirect immunofluorescenee assay. The screened monoclonal antibodies possessed specificity to TGEV. The study provided foundations for research of TGEV pathogenesis and rapid diagnosis.