驴源马疱疹病毒8型ORF72基因原核表达及多克隆抗体制备  

Prokaryotic expression of donkey-derived Equid alphaherpesvirus type 8 ORF72 gene and polyclonal antibody preparation

作　　者：许丹丹 张敬文 张健鹏 王长法 刘文强[1] XU Dandan;ZHANG Jingwen;ZHANG Jianpeng;WANG Changfa;LIU Wenqiang(Liaocheng University,Liaocheng 252000,China)

机构地区：[1]聊城大学,山东聊城252000

出　　处：《黑龙江畜牧兽医》2023年第1期117-121,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：山东省驴产业技术体系项目(SDAIT-27-11);山东省驴产业科技协同创新中心开放课题(319330802);聊城大学理工科研项目(318011701)。

摘　　要：为了获得驴源马疱疹病毒8型ORF72基因编码的糖蛋白D(glycoprotein D,gD)和多克隆抗体,试验根据GenBank上公布的EHV-8 gD基因序列,设计了特异性引物,使用PCR方法扩增得到主要抗原区和跨膜区片段共960 bp,并构建pET-32a-gD960原核表达载体。将重组质粒pET-32a-gD960转化至E.coli BL21(DE3)感受态细胞中,经IPTG诱导并优化表达,获得gD重组蛋白,利用SDS-PAGE和Western-blot分析鉴定表达产物。使用包涵体蛋白纯化试剂盒纯化gD重组蛋白,并免疫小鼠,共免疫3次,每次间隔2周。三免2周后,通过Western-blot对鼠抗EHV-8 gD多克隆抗体进行鉴定,并使用间接ELISA测定其效价。结果表明:试验成功构建EHV-8 gD960基因表达载体并获得gD重组蛋白,gD重组蛋白分子质量约为65 ku,与预期大小相符,且特异性良好。小鼠血清中鼠抗EHV-8 gD960多克隆抗体效价为1∶16 000,该多克隆抗体可以与EHV-8 gD960蛋白发生免疫反应。说明成功表达了EHV-8 gD960重组蛋白并制备了多克隆抗体,多克隆抗体的效价较高,免疫原性较好。In order to obtain glycoprotein D(gD) and polyclonal antibody encoded by ORF72 gene of donkey-derived Equid alphaherpesvirus type 8, based on the sequences of EHV-8 gD gene published on Genbank, specific primers were designed, and the fragments of the main antigenic region and transmembrane region totaling 960 bp were amplified by using PCR method;and the prokaryotic expression vector pET-32a-gD960 was constructed. The recombinant plasmid pET-32a-gD960 was transformed into E. coli BL21(DE3) competent cells, induced by IPTG and optimized for expression to obtain the gD recombinant protein, and the expression products were identified by using SDS-PAGE and Western-blot analysis. The gD recombinant protein was purified by using the inclusion body protein purification kit, and the mice were immunized 3 times, each at 2-week intervals. After 2 weeks after the third immunization, the mouse anti-EHV-8 gD polyclonal antibody was identified by Western-blot and its titer was determined by using indirect ELISA. The results showed that the experiment successfully constructed the EHV-8 gD960 gene expression vector and obtained the gD recombinant protein. The molecular mass of the gD recombinant protein was about 65 ku, which was consistent with the expected size and had good specificity. Mouse anti-EHV-8 gD960 polyclonal antibody had a titer of 1∶16 000 in mouse hyper-immune serum, which was immunoreactive with EHV-8 gD960 protein. The results suggested that the EHV-8 gD960 recombinant protein was successfully expressed and its polyclonal antibody was prepared, which had high titer and good immunogenicity.

关 键 词：驴 马疱疹病毒8型 ORF72基因 原核表达 多克隆抗体 

分 类 号：Q852.659.1[生物学] Q781