安徽农业大学学报2017，Vol.44Issue(3)：373-380,8.DOI:10.13610/j.cnki.1672-352x.20170524.019
基于Iclp的拟态弧菌OmpUAP真核表达重组质粒的构建及其免疫效果初步评价
Construction of Iclp-based recombinant eukaryotic expression plasmid containing the OmpUAP gene from vibrio mimicus and preliminary evaluation on its immune efficacy
摘要
Abstract
In order to investigate if invariant chain-like protein (Iclp) of fish can be used as immune carrier for improving immune efficacy of gene vaccine,the recombinant eukaryotic expression plasmid pcDNA3.1-OmpUAP,which carried the OmpU adhesion function domain gene from Vibrio mimicus,and the plasmid Iclp-based pcDNA3.1-Iclp-OmpUAP were constructed and identified.Healthy Carassius auratus were divided into 4 groups and immunized with pcDNA3.1-OmpUAP,pcDNA3.1-Iclp-OmpUAP,pcDNA3.1 (+) and PBS control.On Day 7,14,21,28 and 35 post-vaccination,the distfbution and transcription of the recombinant plasmids in different tissues of Carassius auratus were detected by PCR and RT-PCR,respectively.The specific antibody against OmpUap and Iclp-OmpUAP in the sera was detected with the triple antibody sandwich ELISA,and then the immuneprotection efficacy was evaluated by challenging V.mimicus at the 35th day of post-vaccination.The results from PCR and RT-PCR indicated that both recombinant plasmids were distributed in the muscle,gill,head kidney,liver and spleen and expressed continuously in different tissues of vaccinated fish within 35 days.Fish immunized with pcDNA3.1-OmpUAP slowly produced low level antibody against OmpUap,and the antibody level at the other detection time points except the 21th day had no significant difference with the empty plasmid control group.Carassius auratus immunized with pcDNA3.1-Iclp-OmpUAP produced specific antibody against [clp-OmpUAP on the 7th day of post-vaccination,and the antibody level continued to rise with prolonging the time,peaked at the 28th day,and then began to decline.The level of antibody against Iclp-OmpUAp was obviously higher than that in pcDNA3.1-OmpUAp group and the empty plasmid control group at the rest of detection time points except the 7th day.The immunprotection rate of fish immunized with pcDNA3.1-Iclp-OmpUAP and pcDNA-OmpUAP could reach 46.7％ and 20.0％ after challenge,respectively,while the fish died completely in the control group of empty plasmid and PBS.Our results suggested that Iclp might be used as immune carrier for improving immune efficacy of gene vaccine in fish.关键词
拟态弧菌/OmpU蛋白/Iclp蛋白/真核重组质粒/免疫效果Key words
Vibrio mimicus/OmpU protein/invariant chain-like protein/recombinant eukaryotic expression plasmid/immune efficacy分类
农业科技引用本文复制引用
刘艳,丁淑荃,陶会竹,曹际,周昊,李槿年..基于Iclp的拟态弧菌OmpUAP真核表达重组质粒的构建及其免疫效果初步评价[J].安徽农业大学学报,2017,44(3):373-380,8.基金项目
国家自然科学基金(31672698)资助. （31672698）
