用人工合成多肽作为半抗原制备Bt Cry1Ac的单克隆抗体  

Preparation of Monoclonal Antibody Against Bt Cry1Ac by Using Synthetic Peptide as Hapten

作　　者：胡小元[1] 张岐蜀[1] 段伟[1] 姜国华[1] 李玲[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2012年第2期88-94,共7页Journal of Agricultural Science and Technology

基　　金：国家转基因生物新品种培育重大专项(2009ZX08012-017B)资助

摘　　要：为了制备Bt Cry1Ac特异性单克隆抗体(MAB),本试验从NCBI获得了Bt Cry1Ac蛋白的氨基酸序列,根据抗原性、亲水性和表位性分析选定Bt Cry1Ac特异性肽段进行人工合成,并将其偶联于匙孔血蓝蛋白(KLH)免疫动物,应用细胞融合技术制备了抗该肽段的杂交瘤细胞34株。通过ELISA和免疫印迹试验从中筛选出与Bt Cry1Ac天然蛋白产生特异性反应的单克隆抗体杂交瘤细胞株一株(6G9)。ELISA和免疫印迹试验结果显示,该克隆株所分泌的MAB能对合成肽和Bt Cry1Ac产生特异性反应,而与同源的Bt Cry1Aa、Bt Cry1Ab天然蛋白均无交叉反应。通过对转基因棉花的ELISA检测结果表明,本试验所制备的Bt Cry1Ac单克隆抗体能够有效的区分常规棉和抗虫棉,并且能对其中的Bt Cry1Ac蛋白进行特异性的识别。In order to prepare monoclonal antibody Bt Cry1Ac,we got the amino acid sequence of Bt Cry1Ac protein from NCBI,and synthesized specific Bt Cry1Ac peptide,according to its antigenicity,hydro-philicity and accessibility on the basis of its structural features by computer assisted analysis.The peptide was linked with carrier keyhole limpet hemocyanin（KHL）,and then used for immunized mouse as antigen.34 strains of hybridoma,which can produce monoclonal antibodies to the peptide,have been obtained by cell fusion technique.One hybridoma strain（6G9）,which was specific to natural Bt Cry1Ac was selected by indirect ELISA and Western blot.The result showed that the monoclonal antibody of hybridoma strain（6G9） can react specifically with the synthetic peptide and Bt Cry1Ac protein,but can not react with Bt Cry1Aa and Bt Cry1Ab proteins by ELISA and Western blot.This result of experiment showed that the monoclonal antibody of hybridoma strain（6G9）can distinguish transgenic Bt CrylAc cotton and general cotton by ELISA,and it can recognize specifically Bt Cry1Ac protein in cotton.

关 键 词：BtCry1Ac 单克隆抗体 合成肽 

分 类 号：Q819[生物学—生物工程]