猪流行性腹泻病毒单克隆抗体的制备及特性分析

为制备猪流行性腹泻病毒(PDEV)的单克隆抗体,并鉴定单克隆抗体特性。本试验采用差速和蔗糖梯度离心纯化PEDV抗原,免疫Balb/c小鼠,将免疫鼠的脾细胞与SP2/0细胞进行融合,经ELISA方法筛选和细胞克隆,得到能分泌鼠抗PEDV单克隆抗体杂交瘤细胞株,制备出相应的单克隆抗体,并分析其特性。试验结果:获得2株能稳定分泌抗PEDV的单克隆抗体,命名为E1和H6株,其中E1单隆抗体为IgG2a亚型,H6单隆抗体为IgM亚型,2株单克隆抗体均具有IFA、ELISA和Western bloting特性。E1杂交瘤细胞株的细胞上清液和腹水的ELISA抗体效价分别26和105,H6杂交瘤细胞株的细胞上清液和腹水的ELISA抗体效价分别24和104。2株单克隆抗体与Vero细胞、猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)均无交叉反应。Western bloting测定结果表明,E1株单克隆抗体能识别PEDV的M蛋白,H6单克隆抗体能识别PEDV的N蛋白。PEDV单克隆抗体的成功研制,为PEDV免疫诊断、表位识别及蛋白研究奠定了良好基础。

Preparation and Characterization of Monoclonal Antibodies Against Porcine Epidemic Diarrhea Virus

This study aimed to obtain the monoclonal antibodies against porcine epidemic diarrhea virus(PEDV) for antibody characterization as well asdisease control.The antigen of PEDV was purifiedby means of differential centrifugation and sucrose gradientcentrifugation.Spleen cells of the Balb/c mice immunized with viral antigen were fused with SP2/0 myeloma cells.Hybridoma cell lines secreting the monoclonal antibodies were isolated by using the indirect ELISA and sub-cloning approach to provide the source for the target antibody.Subsequently,specificities of the antibody were determined by means of IFA,ELISA and the Western blotting.The results obtained the following.(a) Two isolated hybridoma cell lines that could steadily secretethe specific McAbs against PEDV were designated as E1 and H6.(b) The isotyping analysis showed that E1 belonged to IgG2a;and H6,IgM sub-type.The McAbs from them could both bind to the antigen of PED in ELISA,IFA and the western blot analyses.(c) ELISA titers of the supernatants of E1 and H6 were 26 and 24 respectively;while those of the ascites fluids of E1 and H6,105 and 104 respectively.(d) The McAbs from E1 and H6 showed no cross-reactivity with Veto cells,CSFV,TGEV or PRRSV.(e) The Western blotting confirmed that E1 McAbs recognized M protein;and,H6 McAbs,N protein.It appeared that two monoclonal antibodies with a high specificity against PEDV were successfully prepared,and further studies on PEDV immunodiagnosis,epitope discernment and the proteins became possible.