摘要
目的:制备抗鲢鱼主要过敏原小清蛋白的单克隆抗体,并对其基本特性进行鉴定。方法:采用硫酸铵盐析、离子交换柱层析和凝胶过滤柱层析等方法纯化鲢鱼小清蛋白;以纯化的小清蛋白免疫BALB/c小鼠,采用杂交瘤技术制备抗鲢鱼小清蛋白单克隆抗体;通过ELISA法确定抗体亚型;通过腹水诱生法大量制备单克隆抗体;以Protein G Sepharose亲和层析柱纯化制备的单克隆抗体;运用Western blot鉴定单克隆抗体的特异性;以ELISA法分析单克隆抗体的结合位点。结果:从100 g鲢鱼肌肉中可获得约30 mg高纯度小清蛋白;SDS-PAGE结果表明,得到的抗小清蛋白单克隆抗体(A4-C1)纯度较高,亚类为IgG1;Western blot结果显示,A4-C1只与鱼肉粗提物中的小清蛋白产生反应,特异性良好;ELISA分析表明,制备的抗鲢鱼小清蛋白单克隆抗体与商品化抗蛙小清蛋白单克隆抗体(PARV-19)的结合位点可能具有很大部分的重叠,或者存在较大的空间位阻效应。结论:研究中制备的杂交瘤细胞株能稳定分泌高特异性的抗鲢鱼小清蛋白单克隆抗体,可用于后续淡水鱼类小清蛋白检测方法的建立。
Objective：To produce and identify a specific monoclonal antibody against parvalbumin from silver carp（Hypophthalmichthy molitrix） muscle.Methods：Parvalbumin from silver carp muscle was purified by ammonium sulfate fractionation and a series of chromatographies,including DEAE-Sepharose and Sephacryl S-200 HR.Purified parvalbumin was used as antigen to immunize BALB/c mice.Splenic lymphocytes of the immunized mice were fused with SP2/0 myeloma cells and IgG subclass was analyzed by ELISA.Large amount of monoclonal antibody was prepared by injecting immunologically positive cells to mice stomach and harvesting the ascites.The monoclonal antibody was further purified by protein G Sepharose affinity column and the specificity of the antibody was evaluated by Western blot.Epitope comparison of prepared monoclonal antibody with commercial anti-parvalbumin monoclonal antibody was performed by ELISA.Results：Parvalbumin from silver carp was purified to homogeneity with a yield of 30 mg from 100 g of skeletal muscle.The result of SDS-PAGE indicated the high purity of prepared monoclonal antibody（A4-C1）,which belonged to IgG1 subclass.Western blot showed that A4-C1 reacted specifically to parvalbumin in fish muscle crude extracts.The binding epitopes of A4-C1 and commercial anti-parvalbumin antibody（PARV-19） overlapped to a high degree as revealed by ELISA.Conclusion：A strain of hybridoma cell secreting anti-silver carp parvalbumin monoclonal antibody was obtained.This monoclonal antibody with high specificity to fish parvalbumin facilitates further investigation on fish parvalbumins,especially for freshwater fish.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2011年第8期731-734,738,共5页
Chinese Journal of Immunology
基金
国家自然科学基金(20872049
30871947)
福建省科技计划重点项目(2010N0019)
福建省自然科学基金(2010J01044)
李尚大集美大学学科建设基金资助
关键词
小清蛋白
鲢鱼
单克隆抗体
纯化
特异性
Parvalbumin
Silver carp
Monoclonal antibody
Purification
Specificity
