基因Ⅱ型草鱼呼肠孤病毒VP6 N端蛋白原核表达及多克隆抗体制备  

Prokaryotic expression of N-terminal protein of GCRV-ⅡVP6 and preparation of polyclonal antibody

作　　者：李威 吴辉亮 张澳晴 王英英[2] 郑树城[2] 李莹莹[2] 莫绪兵 任燕[2] 潘厚军[2] 尹纪元 施雯 周文礼 王庆[2] LI Wei;WU Huiliang;ZHANG Aoqing;WANG Yingying;ZHENG Shucheng;LI Yingying;MO Xubing;REN Yan;PAN Houjun;YIN Jiyuan;SHI Wen;ZHOU Wenli;WANG Qing(College of Fisheries,Tianjin Agricultural University,Tianjin 300384,China;Pearl River Fisheries Research Institute/Key Laboratory of Fishery Drug Development under the Ministry of Agriculture and Rural Affairs/Guangdong Key Laboratory of Aquatic Animal Immunology and Green Breeding,Chinese Academy of Fishery Sciences,Guangzhou 510380,China;College of Animal Science and Technology,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]天津农学院水产学院,天津300384 [2]中国水产科学研究院珠江水产研究所/农业农村部渔用药物创制重点实验室/广东省水产动物免疫与绿色养殖重点实验室,广州510380 [3]东北农业大学动物科学技术学院,哈尔滨150030

出　　处：《黑龙江畜牧兽医》2025年第4期119-124,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目(2023YFD2400704);国家大宗淡水鱼产业技术体系项目(CARS-45);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金资助项目(2024CG02,2024XT0603,2023TD49);2022年省级乡村振兴战略专项资金种业振兴项目(2022-SPY-00-018);中国水产科学研究院珠江水产研究所基本科研业务费项目(2023SJXT2);广东省农村科技特派员下乡服务专项(KTP20240575)。

摘　　要：为了给基因Ⅱ型草鱼呼肠孤病毒(Grass carp reovirus,GCRV-Ⅱ)诊断方法的建立和相关基因工程疫苗的研制提供材料,试验采用PCR方法扩增了GCRV-ⅡVP6 N端蛋白编码基因,将其克隆至原核表达载体pET-32a(+)中,构建重组表达质粒pET32a(+)-VP6-N,转化至大肠杆菌BL21(DE3)感受态细胞中,构建重组菌pET32a(+)-VP6-N/BL21,经IPTG诱导并优化诱导条件表达GCRV-ⅡVP6 N端蛋白,并对表达的重组蛋白进行可溶性分析,采用Western-blot分析重组蛋白的抗原性,以纯化的重组蛋白免疫新西兰大白兔制备多克隆抗体,采用间接ELISA法测定多克隆抗体效价,采用体外中和试验测定多克隆抗体的体外中和效价。结果表明:扩增出大小为684 bp的GCRV-ⅡVP6 N端蛋白编码基因,构建的重组表达质粒pET32a(+)-VP6-N的PCR鉴定、酶切鉴定结果均与预期大小一致且无碱基突变。重组菌pET32a(+)-VP6-N/BL21诱导后超声破碎沉淀在40 ku处出现特异性条带,与预期蛋白质分子质量大小一致;而上清液未出现明显特异性条带。重组蛋白表达的IPTG最佳诱导浓度为0.6 mmoL/L,最佳诱导时间为6 h。重组蛋白能够与GCRV-Ⅱ阳性草鱼血清发生特异性反应,但与健康草鱼血清不发生反应。所制备多克隆抗体效价为1∶1000000,体外中和效价为1∶40。说明试验成功表达了重组GCRV-ⅡVP6 N端蛋白并制备了多克隆抗体,重组蛋白以包涵体形式存在,具有良好抗原性和免疫原性,制备的多克隆抗体效价较高且具有中和活性。To provide materials for the establishment of diagnostic methods and genetic engineering vaccines for geneⅡ-type Grass carp reovirus(GCRV-Ⅱ),the N-terminal coding gene of GCRV-ⅡVP6 protein was amplified by PCR and cloned into the prokaryotic expression vector pET-32a(+),constructing the recombinant expression plasmid pET32a(+)-VP6-N.This plasmid was then transformed into Escherichia coli BL21(DE3)competent cells to build the recombinant strain pET32a(+)-VP6-N/BL21.The GCRV-ⅡVP6 N-terminal protein was expressed through IPTG induction with optimized conditions,followed by solubility analysis of the recombinant protein.Western blot analysis was then performed to assess the antigenicity of the recombinant protein.New Zealand white rabbits were immunized with the purified recombinant protein to prepare polyclonal antibodies.The neutralizing titer in vitro of the polyclonal antibodies was determined by indirect ELISA and in vitro neutralization test.The results showed that the 684 bp GCRV-ⅡVP6 N-terminal protein coding gene was successfully amplified.PCR and enzyme digestion identification results of the constructed recombinant expression plasmid pET32a(+)-VP6-N matched the expected size with no base mutations.After induction of the recombinant strain pET32a(+)-VP6-N/BL21,a specific band appeared at 40 ku in the ultrasonic disruption precipitate,which was consistent with the expected protein molecular weight size,while no obvious specific band was observed in the supernatant.The optimal IPTG induction concentration for recombinant protein expression was 0.6 mmol/L,and the optimal induction time was 6 h.The recombinant protein could specifically react with the positive grass carp serum of GCRV-Ⅱ,but not with the healthy grass carp serum.The titer of the prepared polyclonal antibody was 1∶1,000,000,and the neutralization titer in vitro was 1∶40.This indicated that the recombinant GCRV-ⅡVP6 N-terminal protein was successfully expressed and polyclonal antibodies were prepared.The recombinant protein exist

关 键 词：草鱼呼肠孤病毒 VP6 N端蛋白 原核表达 多克隆抗体 抗原性 中和活性 

分 类 号：S852.6[农业科学—基础兽医学]