文章摘要
包 林,张 黎,曾晓燕,郭喜玲,史凤娟,黄明明,梁淑仪,汪 华,焦永军.肠道病毒71型VP1蛋白单克隆抗体的制备及初步鉴定[J].南京医科大学学报,2013,(1):37~41
肠道病毒71型VP1蛋白单克隆抗体的制备及初步鉴定
Preparation and characterization of enterovirus 71 VP1 monoclonal antibody
投稿时间:2012-10-10  
DOI:10.7655/NYDXBNS20130108
中文关键词: 肠道病毒71型  VP1蛋白  单克隆抗体
英文关键词: enterovirus 71  VP1 protein  monoclonal antibody
基金项目:国家科技重大专项(2008ZX10002-001,2009ZX10004-904);江苏省医学重点人才项目(RC2011082)
作者单位
包 林 南京医科大学公共卫生学院流行病与卫生统计学系, 江苏 南京 211166 
张 黎 江苏省疾病预防控制中心病原微生物研究所, 江苏 南京 210009 
曾晓燕 江苏省疾病预防控制中心病原微生物研究所, 江苏 南京 210009 
郭喜玲 江苏省疾病预防控制中心病原微生物研究所, 江苏 南京 210009 
史凤娟 江苏省疾病预防控制中心病原微生物研究所, 江苏 南京 210009 
黄明明 南京农业大学动物医学系, 江苏 南京 210095 
梁淑仪 南京医科大学公共卫生学院流行病与卫生统计学系, 江苏 南京 211166 
汪 华 南京医科大学公共卫生学院流行病与卫生统计学系, 江苏 南京 211166 
焦永军 江苏省疾病预防控制中心病原微生物研究所, 江苏 南京 210009 
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中文摘要:
      目的:制备肠道病毒71型(enterovirus 71,EV71)VP1外壳蛋白单克隆抗体。方法:利用重组纯化的EV71-VP1蛋白为抗原免疫BALB/c鼠,按常规杂交瘤技术进行细胞融合,对阳性杂交瘤细胞进行筛选及亚克隆,获得1株能稳定分泌抗EV71-VP1抗体的杂交瘤细胞株6F2B9,细胞体外扩大培养后的上清用Protein G柱纯化,超滤后用BCA法测其浓度,用免疫球蛋白亚类鉴定试剂盒鉴定单克隆抗体亚型,间接ELISA方法检测其效价和特异性,间接免疫荧光法检测其与EV71病毒的特异性结合能力。结果:本研究得到的抗EV71-VP1抗体 DSE-136,纯化后浓度为1.9 g/L,免疫球蛋白亚类为IgG2b,轻链属于κ链;间接ELISA 检测效价为1∶3.2×105;间接免疫荧光结果显示其可与EV71病毒特异性结合。结论:成功制备出1个效价高?特异性好的抗EV71-VP1 单克隆抗体DSE-136,为VP1抗原诊断?疫苗研发及其效果评价奠定了基础。
英文摘要:
      Objective:To obtain an anti-enterovirus 71 (EV71)-VP1 monoclonal antibody, and preliminarily analyze its biological characteristics. Methods:Immunized the BALB/c mice with purified recombinant EV71-VP1 protein. The spleen cells were fused with mouse myeloma cells(Sp2/0). Subsequently, limited dilution method was used to screen positive hybridoma cell lines. After several times of subclone, one hybridoma cell line (6F2B9)was obtained. The supematant of 6F2B9 was collected and purified by affinity chromatography with Protein G sepharose. The concentrations of antibodies were tested by BCA method and its subtype was identified by Ig subtype qualitation kit. The titer and specificity of the McAb were detected by indirect ELISA. We used indirect immunefluorescence assay (IFA) to observe specifical combination with EV71 virus. Results:We succeeded in selecting a hybridoma cell line secreting anti-EV71-VP1 monoclonal antibody. The McAb obtained was named DSE-136 and belonged to IgG2b, κ light chain, and its titer was 1∶3.2×105 and could specifically bind to the surface of EV71 which was detected by IFA. Conclusion:We successfully obtained a mouse anti-EV71-VP1 McAb with high dilution and specificity, which has laid foundation on antigen diagnosis and vaccine research and development for EV71.
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