传染性造血器官坏死病毒糖蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of glycoprotein gene in infectious hematopoietic necrosis virus

通过人工合成方法得到传染性造血器官坏死病毒(IHNV)糖蛋白(G蛋白)基因,将该基因克隆到表达载体pET-30a,构建重组表达质粒pET-30a/IHNV-G,并转化至大肠杆菌BL21(DE3)plySs中。经SDS-PAGE电泳及Western blotting分析表明,在1 mmol/L IPTG(诱导剂)、37℃条件下诱导4 h后,G蛋白在大肠杆菌中成功表达,得到了相对分子质量约为57 000的G蛋白。采用直接切胶免疫小鼠的方法制备多克隆抗体,ELISA分析结果显示,血清效价可达到1∶10 000。本试验中得到的G蛋白和多抗血清可为IHNV疫苗的研制以及胶体金试纸条快速检测方法的建立提供理论基础。

Glycoprotein gene（G） in infectious hematopoietic necrosis virus （IHNV-G） was gained in infectious hematopoietlic tissue through a synthetic method. The full length sequence of IHNV-G gene was synthesized and cloned to prokaryotic expression system pET-30a vector. The plasmid pET-30a/IHNV-G was transformed into BL21 （DE3）plySs in bacterium Escherichia coli. SDS-PAGE and Western blotting showed that the protein was ex- pressed highly as inclusion body, fusion G protein with molecular weight of 57 000. The serum in rabbits four hours immunized with the fusion G protein was then used for ELISA analyses which showed that the serum had titer of 1 ： 10 000, indicating that the G protein and anti-serum could be used in research of IHNV detection method, vaccination and protein function.