摘要
为制备小反刍兽疫病毒(PPRV)非结构蛋白C单克隆抗体,根据PPRV C基因编码多肽链氨基酸序列抗原性分析,设计合成一条含20个氨基酸的抗原肽(CRSGKPRGETPGPLLPEIMQ)和一条含21个氨基酸的筛选抗原多肽(PLRAGERGLAPQAVQHRTLIK),将它们分别与钥孔血蓝蛋白(keyhole limpet hemocyanin,KLH)和生物素羧基蛋白(biotin carboxyl carrier protein,Biotin)交联,制备获得免疫原和筛选抗原.用免疫原肌肉注射5只8～12周龄、体重约20 g的无特定病原体(specific pathogen free,SPF)级BALB/c雌性小鼠,第1次免疫后分别间隔7 d进行第2次免疫和第3次免疫,三免后21 d进行冲击免疫,冲击免疫后第3天采集血液分离血清,采用间接酶联免疫吸附法(ELISA)测得1只免疫小鼠血清抗体效价为1∶312 500,2只为1∶62500.取3只小鼠脾细胞与骨髓瘤细胞SP2/0经聚乙二醇(PEG)融合制备杂交瘤细胞,通过间接ELISA筛选出26株阳性淋巴杂交瘤细胞,进一步经克隆化培养筛选出46株单克隆细胞株.通过Western blot(WB)筛选获得2株能稳定分泌特异性抗PPRV C蛋白单克隆抗体的杂交瘤细胞株,WB、间接免疫荧光(indirect immu-nofluorescence assay,IFA)鉴定显示,制备的单克隆抗体具有较好的灵敏性和病毒反应性.研究结果为进一步阐明C蛋白在PPRV生命周期中的作用奠定了基础,也为小反刍兽疫(PPR)诊断提供了有效的诊断试剂.
Abstract
To prepare monoclonal antibody against non-structural protein C of Peste des petits ruminants virus(PPRV),a 20-amino acids antigen peptide of CRSGKPRGETPGPLLPEIMQ and a 21-amino acid screening antigen peptide of PLRAGERGLAPQAVQHRTLIK were designed and synthesized based on the antigenicity analysis of the a-mino acid sequence of the polypeptide chain encoded by PPRV C gene.The immunogen and screening antigen were prepared by crosslinking with keyhole limpet hemocyanin(KLH)and biotin carboxyl carrier protein(Biotin),re-spectively.Five female specific pathogen free(SPF)grade BALB/c mice aged 8 to 12 weeks and weighing about 20 g were given intramuscular injection of immunogen for the first immunization,while the second immunization and the third immunization were given at intervals of 7 d after the first immunization,the rush immunization was given at 21 d after the third immunization,and the blood was collected and serum was separated on the third day after the rush immunization.The titer of serum antibody of one immunized mouse was 1∶312 500 and the two mice was 1∶62 500 by indirect enzyme linked immunosorbent assay(ELISA)detection.Three mouse spleen cells were fused with mye-loma cells SP2/0 by polyethylene glycol(PEG)to produce hybridoma cells.26 positive lymphohybridoma cells were screened by indirect ELISA,and 46 monoclonal cell lines were screened by clonal culture.Western blot(WB)screened two hybridoma cell lines that could steadily secrete monoclonal antibodies against PPRV C protein.WB and indirect immunofluorescence assay(IFA)showed that the monoclonal antibodies were of good sensitivity and viral re-activity.These results laid a foundation for further elucidating the role of C protein in the life cycle of PPRV,and al-so provided an effective diagnostic reagent for the detection of peste des petits ruminants(PPR).
基金项目
国家自然科学基金(31860696)
中央高校基本科研业务费专项西北民族大学项目(31920190003)
兰州市人才创业创新项目(2020-RC-85)
NETL
NSTL
万方数据