非洲猪瘟病毒VP73蛋白主要抗原表位区的原核表达及多克隆抗体的制备

Prokaryotic Expression of Major Antigenic Epitope Region of African Swine Fever Virus VP73 Protein and Preparation of Polyclonal Antibody

构建重组原核表达载体pET32a-VP73,将pET32a-VP73转化BL21感受态细胞,经IPTG诱导,VP73蛋白主要抗原表位区可稳定高效的表达,SDS-PAGE结果表明,IPTG终浓度为1.0mmol/L,诱导5h蛋白质表达量最高,表达蛋白为融合蛋白,分子质量约为42ku。蛋白质纯化后,经SDS-PAGE及Western blotting鉴定,确定表达产物为非洲猪瘟病毒VP73主要抗原表位区融合蛋白。将纯化的蛋白质免疫新西兰大白兔,免疫前后收集血清。用间接ELISA方法测定血清抗体效价,并以非洲猪瘟阳性血清、兔免疫前后血清及猪瘟、猪繁殖与呼吸综合征、猪伪狂犬病阳性血清为一抗,确定该蛋白质的特异性。结果表明,制备的抗血清效价达到1∶1024000,能与纯化的VP73主要抗原表位区蛋白质发生反应。制备的多克隆抗体为VP73蛋白的免疫学研究和非洲猪瘟血清学诊断奠定了基础。

Constructed recombinant prokaryotic expression vector pET32a-VP73 was transformed into E. coli BL21, major antigenic epitope region of VP73 protein was induced stably and efficiently by IPTG, the result of SDS-PAGE showed, when fi nal concentration of IPTG was 1.0 mmol/I., and pET32a-VP73 was induced 5 h, expression of major antigenic epitope region of VP73 protein was highest, the expressed protein was fusion protein, molecular weight was approximately 42 ku. The pro- tein was identified as major antigenic epitope region of VP73 protein by SDS-PAGE and Western blotting. The purified major antigenic epitope region of VP73 protein was injected to New Zealand rabbits, serum was collected before and after injection. Antibody titer was determined by ELISA. Specificity of this protein was determined by ELISA whose primary antibody were ASF positive serum, rabbits serum before and after injection and positive serum of CSF, PRRS, pig pseudorabies. The result showed the titer of antiserum reached up to 1 ： 1024000, and gave rise to reactions with VP73 major antigenic region protein. The polyclonal antibody built the foundation for immunology research of VP73 and serological diagnosis of African swine fever.