猪细小病毒NS1基因编码蛋白的原核表达及多克隆抗体的制备  

Prokaryotic expression of protein encoded by porcine parvovirus NS1 gene and preparation of polyclonal antibody

作　　者：帅文娜 郭子强 李佳乐 罗梦 李丽薇[1,2] 周艳君 姜一峰[1,2] 童武 童光志[1,2] 李兆龙 高飞[1,2,4] SHUAI Wenna;GUO Ziqiang;LI Jale;UO Meng;LI Liwei;ZHOU Yanjun;JIANG Yifeng;TONC Wu;TONG Guangzhi;LI Zhaolong;GAO Fei(Shanghai Veterinary Research Institute,Chinese Academy ofAgriculural Sciences,Shanghai 200241,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China;College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Shanghai Key Laboratory of Veterinary Biotechnology,Shanghai 200240,China;Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]广西大学动物科学技术学院,广西南宁530004 [4]上海市兽医生物技术重点实验室,上海200240 [5]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《中国兽医科学》2025年第3期314-320,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2022YFD1800800,2021YFD1801401);上海市自然科学基金项目(21ZR1476900);国家自然科学基金项目(32373050,32072861);中国农业科学院科技创新工程项目(CAAS-CSLPDCP-202402);上海市兽医生物技术重点实验室开放基金项目(BD1500010)。

摘　　要：本研究旨在制备PPV-NS1的多克隆抗体,并用猪细小病毒(PPV)和真核质粒pcDNA3.1(+)-NS1-3×Flag进行双重验证。首先将PPV的非结构蛋白编码基因NS1克隆至原核表达载体pETDuet,构建原核质粒pET-PPV-NS1。接着,将原核质粒pET-PPV-NS1转化至感受态E.coli BL21细胞中,经由1 mmol/LIPTG诱导以后,通过SDS-PAGE和Western-blot对NS1表达进行检测,结果表明,NS1基因在感受态细胞中得到了良好的表达。其次,将纯化后获得的NS1蛋白采用多点注射的方式对大白兔进行免疫,制备获得针对重组NS1蛋白的多克隆抗体。然后,将真核质粒pcDNA3.1(+)-NS1-3×Flag转染HEK 293T细胞;同时PPV感染ST细胞后收取蛋白样品,用上述制备的NS1多克隆抗体进行验证。结果显示,制备的NS1多克隆抗体具有良好的反应性和特异性。The aim of this study was to prepare a polyclonal antibody to PPV-NS1 and to perform double validation with porcine parvovirus(PPV)and eukaryotic plasmid pcDNA3.1(+)-NS1-3×Flag.Firstly,the non-structural protein encoding gene NS1 of PPV was cloned into the prokaryotic expression vector pETDuet,and the prokaryotic plasmid pET-PPV-NS1 was constructed.Then,the prokaryotic plasmid pET-PPV-NS1 was transformed into competent E.coli BL21 cells,induced by 1 mmol/L IPTG,and the NS1 expression was detected by SDS-PAGE and Western-blot,and the results showed that the NS1 gene was well expressed in competent cells.Secondly,the purified NS1 protein was injected into rabbits using a multi-point injection method,and polyclonal antibodies against the recombinant NS1 protein were prepared.Then,the eukaryotic plasmid pcDNA3.1(+)-NS1-3XFlag was transfected into HEK 293T cells.At the same time,protein samples were collected after PPV infected ST cells,and the NS1 polyclonal antibody prepared above was used for validation.The results showed that the prepared NS1 polyclonal antibody had good reactivity and specificity.

关 键 词：猪细小病毒 NS1蛋白 多克隆抗体 原核表达 

分 类 号：S852.659.22[农业科学—基础兽医学]