非洲猪瘟病毒p30蛋白单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibody Against African Swine Fever Virus p30 Protein

【目的】制备非洲猪瘟病毒(African swine fever virus,ASFV)p30蛋白单克隆抗体,为ASFV检测方法的研究提供重要试验材料。【方法】应用大肠杆菌表达重组ASFV p30蛋白,并用该蛋白免疫6~8周龄的雌性BALB/c小鼠,应用细胞融合技术将免疫小鼠脾细胞与SP2/0细胞融合;通过间接ELISA方法筛选分泌p30蛋白特异性抗体的阳性杂交瘤细胞,腹腔接种小鼠制备腹水抗体;应用ELISA方法鉴定单克隆抗体的类/亚类,通过Western blotting和间接免疫荧光试验(IFA)鉴定单克隆抗体与p30-N端(1―105位氨基酸)和p30-C端(100―194位氨基酸)区域的反应活性;针对抗体识别的p30蛋白区域合成不同肽段,通过ELISA和斑点免疫印迹法鉴定抗体识别的抗原表位。应用间接ELISA方法分析单克隆抗体与ASFV p72蛋白、猪圆环病毒2型(PCV2)衣壳(Cap)蛋白、猪流行性腹泻病毒(PEDV)S蛋白的交叉反应性,以及ASFV抗血清对单克隆抗体抗原结合活性的阻断作用。【结果】获得了2株杂交瘤细胞(C7和G10),其分泌的单克隆抗体(C7和G10)都属于IgG1亚类和κ型(IgG1κ),杂交瘤细胞培养上清和腹水内C7和G10的抗体效价分别为1∶1280、1∶640与1∶10^(7)、1∶10^(6);2株抗体均与p30-C端(100―194位氨基酸)区域特异性结合,并识别同一个抗原表位115 CTSSFETLFEQEPSSEVPKD^(134);2株抗体与ASFV p72蛋白、PCV2 Cap蛋白及PEDV S蛋白无交叉反应;ASFV抗血清能有效阻断2株单克隆抗体与p30蛋白结合。【结论】获得2株分泌p30单克隆抗体的杂交瘤细胞株,鉴定了单克隆抗体的抗原识别表位,丰富了p30蛋白的抗原表位信息,为ASFV致病机制的进一步研究提供支撑。

【Objective】To provide important experimental materials for the research of African swine fever virus(ASFV)detection method,the monoclonal antibody against p30 protein of ASFV was prepared.【Method】Recombinant ASFV p30 protein was expressed by Escherichia coli and used to immunize female BALB/c mice aged 6-8 weeks.The splenocytes of the immunized mice were fused with SP2/0 cells by cell fusion technique.The positive hybridoma cells secreting specific antibody against p30 protein were screened by indirect ELISA,and the hybridoma cells were intraperitoneally inoculated into mice to produce ascites antibodies.The class/subclass of the monoclonal antibodies were identified by ELISA,and the reactivity of the monoclonal antibody with p30-N terminal(1-105 amino acids)and p30-C terminal(100-194 amino acids)of p30 protein were identified by Western blotting and indirect immunofluorescence assay(IFA).Different peptides were synthesized for the p30 protein region recognized by the antibody,and the epitopes recognized by antibodies were identified by ELISA and Dot blotting.Indirect ELISA was used to analyze the cross-reactivity of the monoclonal antibodies with ASFV p72 protein,Porcine circovirus type 2(PCV2)capsid(Cap)protein,Porcine epidemic diarrhea virus(PEDV)S protein,and the blocking effect of ASFV antiserum on the antigen binding activity of monoclonal antibodies.【Result】Two hybridoma cells(C7 and G10)were obtained,and the monoclonal antibodies(C7 and G10)secreted by the two cell lines belonged to the IgG1 subclass andκtype(IgG1κ).The titers of C7 and G10 in hybridoma cell culture supernatant and ascites were 1∶1280,1∶640,and 1∶10^(7),1∶10^(6),respectively.The two monoclonal antibodies were specifically bound to the p30-C terminal(100-194 amino acids)region,and recognized the same epitope 115 CTSSFETLFEQEPSSEVPKD ^(134).There was no cross reaction with ASFV p72,PCV2 Cap and PEDV S proteins,and anti-ASFV positive serum could effectively block the two monoclonal antibodies from binding to p30 protein.【Conclusion】Two hybridoma cell lines secreting p30 monoclonal antibody were obtained,and the epitopes recognized by monoclonal antibodies were identified,which enriched the information of the epitopes of p30 protein,and laid an foundation for further research on the pathogenesis of ASFV.