鸡传染性法氏囊病病毒VP2与白喉毒素截短体DT390融合蛋白的真核表达及免疫原性检测  

Eukaryotic Expression and Immunogenicity Detection of Fusion Protein of Chicken Infectious Bursal Disease Virus VP2 and Truncated Diphtheria Toxin DT390

作　　者：孟维金 范阔海[2] 常熊熊 尹伟[1] 李宏全[1] 王志瑞[1] MENG Weijin;FAN Kuohai;CHANG Xiongxiong;YIN Wei;LI Hongquan;WANG Zhirui(Clinical Veterinary Laboratory,College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;Laboratory Animal Management Center of Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学动物医学学院,临床兽医学实验室,太谷030801 [2]山西农业大学实验动物管理中心,太谷030801

出　　处：《中国畜牧兽医》2021年第12期4652-4660,共9页China Animal Husbandry & Veterinary Medicine

基　　金：山西省重点研发计划项目(201903D421037)。

摘　　要：为探究白喉毒素截短体DT390是否对鸡传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)VP2蛋白具有免疫增强作用,本研究利用有白喉毒素抗性的毕赤酵母表达系统对DT390和VP2进行融合表达,用SDS-PAGE和Western blotting方法对表达的融合蛋白DT390-VP2进行了鉴定。将24只21日龄SPF雏鸡随机均分为4组:VP2组、DT390-VP2组、PBS(对照组)和B87组(阳性对照组),在第0和14天,分别对各组雏鸡进行免疫,在首免后第28天,每只雏鸡接种100 BID_(50)的IBDV BC6/85毒株,采用ELISA法检测免疫前后不同时间各组雏鸡血清的VP2特异性抗体水平,采用法氏囊组织切片HE染色及病理损伤评分来评价法氏囊的损伤程度,评价融合蛋白DT390-VP2在雏鸡体内的免疫原性和保护功能。SDS-PAGE和Western blotting分析结果显示,表达和提纯后的目的蛋白确为糖基化的融合蛋白DT390-VP2(91.26 ku)。体内试验结果显示,首次免疫21 d后,DT390-VP2组的VP2特异性抗体滴度分别显著和极显著高于B87组(P<0.05)和VP2组(P<0.01);首次免疫28 d后,DT390-VP2组的VP2特异性抗体滴度极显著高于VP2组(P<0.01);与VP2蛋白相比,融合蛋白DT390-VP2减轻了雏鸡法氏囊淋巴滤泡的萎缩程度。综上所述,白喉毒素截短体DT390以融合表达的方式增强了VP2的体液免疫原性,融合蛋白DT390-VP2在一定程度上减轻了IBDV BC6/85毒株造成的组织损伤。In order to explore whether truncated diphtheria toxin DT390 had immune enhancing effect on VP2 protein of Infectious bursal disease virus(IBDV),in this study,DT390 and VP2 were fused and expressed by Pichia pastoris expression system with diphtheria toxin resistance.The expressed fusion protein DT390-VP2 was identified by SDS-PAGE and Western blotting.Twenty four 21 days old SPF chicks were randomly divided into four groups:VP2 group,DT390-VP2 group,PBS(control group)and B87 group(positive control group).The chicks in each group were immunized on the the 0 and 14th days,respectively.On the 28th day after the first immunization,each chick was inoculated with IBDV BC6/85 strain of 100 BID_(50).The serum VP2 specific antibody levels of chicks in each group at different time before and after immunization were detected by ELISA.HE staining and pathological damage score were used to evaluate the damage degree of Fabricius,and the immunogenicity and protective function of fusion protein DT390-VP2 in chicks.SDS-PAGE and Western blotting analysis showed that the expressed and purified target protein was glycosylated fusion protein DT390-VP2(91.26 ku).The results of in vivo test showed that 21 days after the first immunization,the titer of VP2 specific antibody in DT390-VP2 group was significantly and extremely significantly higher than that in B87 group(P<0.05)and VP2 group(P<0.01),respectively.28 days after the first immunization,the titer of VP2 specific antibody in DT390-VP2 group was extremely significantly higher than that in VP2 group(P<0.01).Compared with VP2 protein,the fusion protein DT390-VP2 reduced the atrophy of bursal lymph follicles in chicks.In conclusion,truncated diphtheria toxin DT390 enhanced the humoral immunogenicity of VP2 by fusion expression,the fusion protein DT390-VP2 reduced the damage of tissue caused by IBDV BC6/85 strain to a certain extent.

关 键 词：鸡传染性法氏囊病病毒(IBDV) VP2蛋白 DT390 免疫原性 

分 类 号：S852.657[农业科学—基础兽医学]