用合成多肽为半抗原制备Bt Cry1Ab的单克隆抗体  

Preparation of the Monoclonal Antibody Against Bt Cry1Ab 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：《农业生物技术学报》2013年第4期475-482,共8页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2009ZX08012-017B);中国农业科学院生物技术研究所基本科研业务费专项资金(NO.2011-13)

摘　　要：由于Bt Cry 1Aa、Cry 1Ab和Cry 1Ac晶体蛋白之间具有很高的同源性（82％～90％），采用常规的单抗制备方法很难制取特异性强的Bt Cry1Ab单抗，为了制备抗Bt Cry1Ab蛋白的特异性单克隆抗体（MAB），本研究从NCBI获得了Bt Cry 1Ab蛋白的氨基酸序列，根据ANTHEPORT和DNAStar软件对其抗原性、亲水性和表位性分析结果选定Bt Cry1Ab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22株。通过ELISA试验从中筛选出与Bt Cry1Ab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（3A10）。经检测，其分泌的抗体亚类为IgGl型：轻链属K型；杂交瘤细胞株染色体数目为89～108条；用其制作的腹水对BtCrylAb合成肽的反应效价为1：1×10^7；对Bt Cry1Ab天然蛋白的反应效价为1：1×10^4；纯化后的抗体对Bt Cry1Ab合成肽的效价为1：1×10^8；对Bt Cry1Ab天然蛋白的效价为1：2×10^4。抗体的相对亲和力为0．5μg／mL，对Bt Cry1Ab蛋白的最低可检测值为10ng／mL。ELISA结果显示，3A10杂交瘤细胞株所分泌的MAB能特异性识别合成肽和Bt Cry1Ab蛋白，而对同源的Cry1Ac和Cry1Aa蛋白无交叉反应：本研究所制备的Bt Cry1Ab单克隆抗体能够对常规棉和抗虫棉（Gossypium hirsutum L．）进行有效的区分，并且能特异性的识别其中的Bt Cry1Ab蛋白。Because Bt Cry 1Aa, Cry lAb and Cry 1Ac share high sequence identity（82%-90%）, it is difficult to prepare Bt CrylAb monoclonal antibody that has highly specific by using conventional method. In order to prepare monoclonal antibody against Bt CrylAb, we acquired the amino acid sequence of Bt CrylAb protein from NCBI. According to the antigenicity, hydrophilicity and accessibility analyzed results by ANTHEPORT and DNAStar computer software, the specific peptide of Bt CrylAb was synthesized by a chemical process. The Bt CrylAb peptide was linked with carrier keyhole limpet hemocyanin（KHL）, and then used for immunized mouse as antigen. Twenty-two strains of hybridoma, which produced monoclonal antibodies to the peptide, had been obtained by cell fusion technique. One hybridoma strain（3A10）, which was specific to natural Bt CrylAb selected by indirect ELISA. The result of isotype analysis showed that the monoclonal antibody of 3A10 hybridoma strain belong to IgG1 subclass and the light chain was K chain. The hybridoma chromosome number was 89-108. The monoclonal antibody of hybridoma ascites titers to synthetic peptide was 1：1×10^7 and to natural Bt CrylAb protein was 1：1×10^4. The purified MAB titers to synthetic peptide was 1：1×10^8 and to natural Bt CrylAb protein was 1：2×10^4. The relative affinity of the monoclonal antibody was 0.5 μg/mL. The monoclonal antibody of hybridoma strain could react specifically with the Bt CrylAb protein without reacting with Bt CrylAc and Bt CrylAa proteins by ELISA. The lowest detectable value of the monoclonal antibody to Bt CrylAb was 10 ng/mL by indirect ELISA detection. The monoclonal antibody of hybridoma strain can distinguish transgenic Bt CrylAb cotton and general cotton（Gossypium hirsutum L.） by ELISA, and it can recognize specifically Bt CrylAb protein in cotton.

关 键 词：BT CRY1AB 单克隆抗体 合成肽 

分 类 号：S154[农业科学—土壤学]