牛乳腺炎标志物血清淀粉样蛋白A3的原核表达及其单克隆抗体的制备

Preparation of monoclonal antibody against bovine mastitis biomarker serum amyloid A3 expressed by E.coli

为了表达牛乳腺相关血清淀粉样蛋白A3(M-SAA3)基因,获得其重组蛋白及单克隆抗体(MAb),本研究将编码M-SAA3的基因片段经密码子优化后接入载体pET-30a(+)中,然后转入大肠杆菌BL21(DE3)感受态细胞中进行表达。以镍柱纯化蛋白后,免疫BALB/c小鼠制备M-SAA3 MAb。结果显示,扩增的M-SAA3基因序列无误;M-SAA3基因成功转入表达载体;菌液的诱导表达D600值=0.8为最优条件;重组蛋白纯度可达98%,浓度为1.146 mg/m L;制得7株MAb效价均>1∶1600,亚型均为Ig G3,研制的3B2B4 MAb可分别与M-SAA3的重组及天然蛋白特异性结合。该成果为将来针对M-SAA3的奶牛乳腺炎检测产品研发提供了重要原料。

The aim of this study was to obtain recombinant bovine mammary-associated serum amyloid protein A3(M-SAA3)and its monoclonal antibody(MAb).After codon bias optimization for E.coli,the synthesized gene fragment encoding M-SAA3 was cloned into the expression vector p ET-30 a(+).Then it was transformed into E.coli BL21(DE3)competent cells for protein expression.The protein was purified by nickel chromatography,and the monoclonal antibody was prepared by immunizing BALB/c mice with the obtained recombinant protein.The sequencing showed that M-SAA3 gene was successfully cloned into the expression vector.The optimal induced expression condition of bacterial liquid was a D600 of 0.8.Purity of the prepared protein was up to 98%,and the concentration was 1.146 mg/m L.The titer of all 7 strains of MAb were>1∶1600,and the subtype were all Ig G3.Among them,the 3 B2 B4 MAb could specifically bind to M-SAA3 recombinant and natural protein,respectively.This study provided important raw materials for the future research on the mastitis immunoassay of bovine M-SAA3.