羊肠道病毒单克隆抗体的制备及鉴定

Generation and characterization of monoclonal antibody against glycoprotein VP1 encoded by caprine enterovirus CEV-JL14

根据本实验室分离的国内首株羊肠道病毒CEV-JL14的核苷酸基因组序列,设计并合成了羊肠道病毒VP1基因的引物,并应用RT-PCR扩增了VP1基因片段。扩增的VP1基因片段克隆到原核表达载体pGEX-4T-1的EcoRⅠ/BamHⅠ的酶切位点。重组质粒经酶切鉴定和序列测定正确后,转化到BL21,并以IPTG诱导表达了VP1重组蛋白。重组蛋白纯化后以弗氏完全佐剂乳化,按一定的程序免疫BALB/c小鼠,加强免疫3次后,取小鼠脾细胞与骨髓瘤细胞融合,经过3次亚克隆获得了5株表达VP1单克隆抗体的杂交瘤细胞株。以ELISA对杂交瘤分泌的IgG亚类进行了鉴定,结果4株为IgG1亚类,1株为IgG2b。以免疫酶单层细胞技术鉴定了单克隆抗体,结果显示,获得的单克隆抗体可以特异性检出羊肠道病毒抗原。本试验在国内首次获得了抗羊肠道病毒的单克隆抗体,将为羊肠道病毒的致病机理、诊断及流行病学研究奠定基础。

VP1 gene encoded by the newly identified caprine enterovirus CEV-JL 14 was amplified and cloned to prokaryotic expression vector pGEX-4T-1. Recombinant GST-VP1 fusion protein was expressed, purified, emulsified with Freund＇s complete adjuvant and used to immunize the BALB/c mice following a standard procedure. The spleen cells from immunized mice were collect- ed and fused with myeloma cells after its antibody titer reached over 104 times detected by indirect ELISA. Hybridoma cell clones secreting monoclonal antibodies against VP1 were screened and their stability and specificity were further determined. The identified hybridoma cells were injected to mice intraperitoneally and ascites were collected at 7 DPI. Isotypes of the monoclonal antibodies against the recombinant VP1 protein were characterized to be either IgG1 or IgG2b,which showed a high specificity for detection of caprine enterovirus antigens by immunoperoxidase monolayer as- say,thus laying a solid basis for future study related to viral pathogenesis,detection and diagnostics for caprine enterovirus infection.