【摘要】 利用已构建好的pET-30a重组菌E.coli Rosetta(DE3)表达鹅细小病毒(GPV)VP3重组蛋白,经Ni-NTA亲和层析纯化后作为免疫原,经腹膜腔接种4～6周龄BALB/c小鼠3次,末次免疫后第3d取小鼠脾细胞与SP2/0骨髓瘤细胞进行融合。用建立的间接ELISA方法进行筛选,经4次亚克隆,最终获得4株能稳定分泌抗GPVVP3单克隆抗体的杂交瘤细胞株,分别命名为1F1、2A9、3B11和4A2。亚类鉴定结果显示,单抗1F1为IgG2b亚型,其余3株单抗为IgG1亚型,轻链均为κ链。经间接ELISA检测,单抗1F1、2A9、3B11和4A2的腹水效价分别为1:819200、1:1638400、1:409600和1:819200。Western-blot分析表明,获得的4株单克隆抗体均能特异性识别GPVVP3重组蛋白;间接免疫荧光试验证明,这4株单抗能够与天然GPV结合。更多还原

【Abstract】 The goose parvovirus(GPV) VP3 protein expressed in the recombinant E.coli Rosetta(DE3) was used as immunogen after purification by Ni-NTA.The 4 to 6 weeks-old BALB/c mice were intraperitoneally immunized with the VP3 protein for three times,then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice on day 3 post-last-immunization.Four hybridoma cell lines against the VP3 protein were obtained by screening with the indirect ELISA,named 1F1,2A9,3B11 and 4A2,respectively.The first monoclonal antibody(McAb) were identified to be IgG2b,and the others to be IgG1 with κ light chain.The ELISA titer of the 4 McAbs ascites were 1∶819 200,1∶1 638 400,1∶409 600 and 1∶819 200,respectively.Western-blot analysis showed that the four McAbs could react with the GPV recombinant VP3 protein specifically.IFA showed that the four McAbs could combine with the natural GPV.更多还原