锦鲤疱疹病毒ORF1基因的克隆、表达及多克隆抗体的制备

PCR Amplification Prokaryotic Expression and Polyclonal Antibody Preparation of Koi Herpesvirus Virus ORF1 Gene

为了进行锦鲤疱疹病毒(Koi Herpesvirus Virus,KHV)诊断方法、疫苗研制和蛋白功能研究。通过选择基因保守性极强的ORF1基因作为研究对象,设计1对特异性引物进行PCR克隆。目的基因与表达载体PET构建重组质粒,将重组质粒转化大肠杆菌E.coliBL21(DE3)后诱导表达,再将成功表达的目的蛋白免疫小鼠制备多克隆抗体。PCR产物经电泳显示,所扩增的基因长度774bp,与目的基因长度相符。蛋白表达产物经SDS-PAGE和Western-bolt检测,重组表达质粒K1-PET成功诱导表达,表达蛋白为37.3KD,为包涵体。免疫小鼠获得多克隆抗体,经酶联免疫检测发现其效价达到1:27000。表明表达出的目的蛋白具有免疫原性,实验获得的表达蛋白和多抗血清为KHV的疫苗研制和免疫学检测方法的建立奠定了基础。

Koi herpesvirus （KHV） was an emerging pathogen causing mass mortality in Koi. The conserved ORF1 gene of KHV was selected to amplification by PCR. The full length of this gene was 774 bp encoding an predicted envelope protein VP1 which consisted of 258 amino acids. The amplified gene was subcloned into pET-32a vector to construct recombinant expression plasmids named K1-PET, which then was transformed into E.coli BL21 （DE3） and expressed by IPTG inducement. SDS-PAGE and Western blot results showed that K1-PET can be expressed highly. The molecular weight of fusion VP1 protein was 37.3 KD, and the protein was mainly expressed as inclusion body. The rabbit serum obtained after four immunizations with the fusion VP1 protein, was then used for ELISA analyses. The results showed that the potency of antiserum was 1： 27000, and the VP1 protein had high immunogenicity. Therefore, the VP1 protein and antiserum could be used in research of KHV detection method, vaccine produce and protein function study.