猪伪狂犬病毒gE蛋白的酵母表达及其单克隆抗体的筛选

Expression of Pseudorabies Virus gE Protein in Pichia pastoris and Screening of Its Monoclonal Antibodies

为获得具有生物学活性的猪伪狂犬病毒(PRV)gE蛋白及其特异性单克隆抗体,利用电转技术将pPICZαA-gE重组质粒转入毕赤酵母X-33并筛选出阳性克隆,通过SDS-PAGE、Western blot鉴定筛选出PRV gE蛋白表达菌株。通过诱导表达获得gE分泌蛋白,并利用Ni柱进行纯化,应用Western blot、Dot blot、ELISA方法对纯化蛋白进行活性鉴定。同时将纯化蛋白免疫BALB/c小鼠,细胞融合后通过免疫过氧化物酶单层细胞试验(IPMA)筛选单克隆抗体,并对单克隆抗体进行了反应性测定、效价测定及亚型鉴定。结果显示,PRV gE蛋白在毕赤酵母中成功表达,并获得了纯化蛋白,纯度达90%以上,且具有良好的生物学活性,与PRV阳性血清反应性良好;筛选到4株敏感性强、特异性良好的gE单克隆抗体,均可同时特异识别PRV gE蛋白及病毒,分别命名为2F10、4C10、9E1、10C3。综上,用酵母系统成功表达了PRV gE蛋白,并筛选到4株针对PRV gE蛋白的单克隆抗体。

In order to obtain pseudorabies virus(PRV)gE protein with biological activity and its specific monoclonal antibodies,in this study,the recombinant plasmid pPICZαA-gE was transformed into Pichia pastoris X-33 by electroporation,and the positive clones were screened.PRV gE protein expression strains were screened by SDS-PAGE and Western blot.The gE secretory protein was obtained by inducing expression and purified by Ni column.The purified protein was identified by Western blot,Dot blot and ELISA.At the same time,BALB/c mice were immunized with purified protein.After cell fusion,monoclonal antibodies were screened by immunoperoxidase monolayer assay(IPMA)and the reactiveness,titer and subtype of monoclonal antibodies were identified.The results showed that PRV gE protein was successfully expressed in Pichia pastoris and purified by Ni column.The purity of PRV gE protein was over 90%,and the protein had good biological activity and well reactivity with PRV positive serum.Four strains of monoclonal antibodies of gE with high sensitivity and specificity were screened,which could recognize PRV gE protein and virus specifically at the same time.They were named 2F10,4C10,9E1 and 10C3 respectively.In conclusion,PRV gE protein was successfully expressed in yeast system,and four strains of monoclonal antibodies against PRV gE protein were screened.