木薯钙调蛋白的原核表达及其单克隆抗体的制备

钙调蛋白(CaM)作为重要的抗逆信号转导蛋白,在调控木薯抗逆境和块根采后生理腐烂中起重要作用,为给快速检测木薯在不同生长环境中CaM蛋白的表达水平提供优良抗体,克隆木薯CaM基因并将目的基因插入原核表达载体,经Escherichia coli表达并纯化,用纯化的融合蛋白ACP-CaM免疫Balb/C小鼠,间接ELISA测定小鼠血清效价后,取小鼠脾细胞与SP2/0细胞融合,筛选能产生抗CaM单克隆抗体的杂交瘤细胞株,用Western blot、ELISA等方法对制备的单克隆抗体进行初步鉴定。Western blot分析结果显示原核表达重组质粒在E.coli中能高效表达CaM,免疫小鼠后取效价高的2#小鼠脾细胞和SP2/0细胞融合,共获得7株效价均达到106以上、能稳定分泌抗CaM抗体的细胞株,这7株单抗与淀粉磷酸化酶、His、BSA均无交叉反应,4D5株与标签蛋白ACP有交叉反应,表明其余6株均为CaM特异性抗体;抗体亚型鉴定结果显示7株单抗均为IgG型抗体。

Prokaryotic Expression and Monoclonal Antibody Preparation of Calmodulin Originated from Cassava

Calmodulin(CaM) is an important signal-transduction protein. It will play an important role in regulating tolerance to extreme conditions and postharvest physiology deterioration. In order to provide good antibody in rapid determination of CaM expression in cassava growing at different environments,in the present study,cDNA of cassava CaM was cloned into the prokaryotic expression vector,CaM was expressed in E.coli and then purified. The purified fusion protein ACP-CaM was used to immunize Balb/c mice and the titer of mice serum was determined by indirect ELISA. The spleen lymphocyte was fused with SP2/0 hybridoma to produce monoclonal antibody against CaM. Western blot and ELISA were carried out to preliminarily identify the monoclonal antibody produced by these hybridomas. Western blot result showed that recombinant plasmid in E.coli can expressed CaM efficiently. SP2/0 cells and spleen cells of 2# mice were fused,then 7 strains,which antibody titer was more than 10⁶,were received,no cross reaction with starch phosphorylase,His and bovine serum albumin. 4D5 strains has cross reaction with carrier protein ACP,6 strains of CaM were specificity antibodies. All of strains were IgG type.