鳜传染性脾肾坏死病毒ORF093基因的克隆、表达及其重组蛋白的免疫原性分析

从患传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus,ISKNV)病的鳜体内获得ORF093基因。采用PCR方法扩增ISKNV ORF093基因全长,克隆入原核表达载体pET32a(+),IPTG诱导表达,Ni柱纯化后,免疫新西兰大白兔制备兔抗重组093蛋白血清,免疫印记分析兔抗血清的特异性,中和实验和免疫保护实验分析重组093蛋白的免疫原性。结果显示:ISKNV重组093蛋白可以在大肠杆菌中以包涵体形式存在;制备的兔抗血清可以特异性识别重组093蛋白,对ISKNV具有中和作用,可给鱼体提供至少10 d的100%预防保护;重组093蛋白对ISKNV的攻击具有保护作用,攻毒后15 d,093免疫组平均相对保护率为45.3%。结果说明重组093蛋白具有一定的免疫原性,可作为ISKNV候选疫苗抗原和治疗血清制备抗原。本研究通过中和实验及免疫保护实验对重组093蛋白的免疫原性进行分析,旨在为鳜ISKNV重组亚单位疫苗的研制奠定基础。

Cloning and expression of ORF093 gene and the immune effect of its recombinant protein of infectious spleen and kidney necrosis virus in Siniperca chuatsi

The full-length ORF093 gene from infectious spleen and kidney necrosis virus (ISKNV) isolated from mandarinfish, Siniperca chuatsi, was amplified by PCR, and then cloned into the prokaryotic expression plasmid pET32a(+). The recombinant 093 protein expression was induced by IPTG and purified by Ni-NTA affinity chromatograph. Polyclonal antibodies were raised in rabbits against the purified protein and the reaction of the antibody was confirmed by western blotting using the purified protein and the spleen and kidney of healthy and diseased mandarin fish. The immunogenicity of 093 protein was investigated by neutralization test and immune protection test. The results showed that the recombinant 093 protein was found as the inclusion bodies in E. coli. Western blotting of rabbit sera against recombinant 093 protein and the spleen and kidney of diseased mandarin fish showed a protein recognition. The antiserum could neutralize ISKNV infection and could provide fish 10-days-long 100% protection. The recombinant 093 protein could protect mandarinfish against virulent challenge with ISKNV and the average RPS of 093 group was 45.3%. The results indicated that the recombinant 093 protein had similar antigenicity to original 093 protein of ISKNV. It could be a potential vaccine candidate and antigen candidate for antiserum treatment.