鸭瘟病毒UL6基因的原核表达及多克隆抗体的制备

为制备鼠抗鸭瘟病毒(DPV)UL6基因的多克隆抗体,采用PCR方法扩增出UL6基因,连接原核表达载体p ET-32a(+),构建表达质粒p ET-UL6,并将其转化大肠杆菌BL21(DE3),于37℃经1.0 mmol/L的IPTG诱导4 h,SDS-PAGE检测融合蛋白的表达情况,将目的蛋白免疫Balb/c小鼠,利用ELISA方法检测特异性抗体效价。结果显示,构建的原核表达质粒经IPTG诱导能正确表达,且表达的重组蛋白能与DPV阳性血清反应,制备的多克隆抗体效价可达1∶16 000。表明,制备的多克隆抗体具有良好的免疫原性,可以用于UL6基因的表达检测。

Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Plague Virus UL6 Gene

To prepare mouse anti-duck UL6 of duck plague virus polyclonal antibodies,a pair of specific primers were designed,UL6 gene was amplified by PCR,the fragment of the UL6 gene was cloned into the prokaryotic expression vector pET-32a( +),the resultant recombinant plasmid pET-UL6 was constructed and transformed into E. coli BL21(DE3). Optimally expressed under the induction of 1. 0 mmol/L IPTG at 37 ℃ for 4 hours. SDS-PAGE analysis showed that the UL6 protein was successfully expressed, and it could be recognized by DPV positive serum. After Westernblot identification,the antiserum against UL6 protein was produced by immunizing Balb/c mouse with recombinant protein. ELISA results showed that the antibodies titer was 1∶16 000. In this research,UL6 gene was successfully expressd in E. coli,the immunogenicity of the polyclonal antibodies was good,and the antiserum could be used for detection of UL6 gene expression.