猪圆环病毒2型病毒样颗粒的制备及免疫原性评价  

Preparation and immunogenicity evaluation of porcine circovirus type 2 virus-like particles

作　　者：赵强[1,2] 赵云环 郭禹 翟刚 张帅 郭晓旭[1] 范京惠 左玉柱[1,2] ZHAO Qiang;ZHAO Yunhuan;GUO Yu;ZHAI Gang;ZHANG Shuai;GUO Xiaoxu;FAN Jinghui;ZUO Yuzhu(College of Veterinary Medicine,Hebei Agricultural University,Baoding,Hebei 071001,China;Hebei Veterinary Biotechnology Innovation Center,Baoding,Hebei 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001

出　　处：《中国兽医学报》2022年第8期1529-1534,共6页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划资助项目(19226622D);河北省农业产业技术体系生猪创新团队资助项目(HBCT2018110207)。

摘　　要：旨在利用原核表达系统在大肠杆菌中制备出猪圆环病毒2型(PCV2)病毒样颗粒(VLP),用改良的镍亲和层析法纯化并在小鼠体内评价其免疫原性。通过PCR方法从发病猪中扩增出PCV2的ORF2序列,对其全长进行密码子优化并交由公司合成重组质粒pET-32a-yCap;以BL21(DE3)为表达菌株,经IPTG诱导表达,利用SDS-PAGE与Western blot对Cap蛋白的表达方式及抗原特异性进行鉴定;利用改良的镍亲和层析法纯化带有His-Tag标签的重组蛋白;利用透射电镜观察所纯化的载体融合蛋白能否形成VLP;用自制的VLP疫苗与PCV2灭活疫苗(ZJ/C株)分别免疫小鼠,利用商业化PCV2 IgG抗体ELISA检测试剂盒评价其免疫原性。结果显示,成功得到PCV2 ORF2序列和重组质粒pET-32a-yCap,重组质粒在大肠杆菌中主要以可溶性蛋白形式表达,蛋白大小为47 kDa;经镍亲和层析法纯化可得到单一的目的蛋白;重组Cap蛋白能与PCV2多克隆抗体及HRP标记的6*His,His-Tag小鼠单克隆抗体发生特异性结合;经过磷钨酸负染色的蛋白悬液在透射电镜下可观察到大量规则的、直径在17~20 nm的VLP;2种疫苗均能诱导刺激小鼠产生抗体应答反应,VLP组诱导产生的水平更高。综上所述,本研究实现了PCV2 Cap蛋白在大肠杆菌中的可溶性表达,经改良的镍亲和层析法可得到单一的目的蛋白,能够在体外自发的组装成病毒样颗粒,免疫小鼠后可刺激抗体应答反应,可用于制备亚单位疫苗及相关生物制品。This study aims to prepare virus-like particles(VLP)of porcine circovirus type 2(PCV2)in E.coli using a prokaryotic expression system,and to purify it by improved nickel affinity chromatography and evaluate its immunogenity in mice.The ORF2 sequence of PCV2 was amplified from the diseased pigs by PCR,and the sequence was optimized for codons and the recombinant plasmid pET32 a-yCap was synthesized.BL21(DE3)was used as the expression strain,and the expression was induced by IPTG.SDS-PAGE and Western blot were used to identify the protein expression and antigen specificity of Cap protein.Improved nickel affinity chromatography was used to purify recombinant protein with His-tag.Transmission electron microscope was used to observe whether the purified carrier fusion protein could form VLP.Mice were immunized with self-made VLP vaccine and PCV2 inactivated vaccine(ZJ/C strain)separately,and the immunogenicity was evaluated with commercial PCV2 IgG antibody ELISA test kit.The results showed that the PCV2 ORF2 sequence and the recombinant plasmid pET32 a-yCap were successfully obtained.The recombinant plasmid was mainly expressed as a soluble protein in E.coli,with a protein size of47kDa.Target protein could be obtained after purification by nickel affinity chromatography.Recombinant Cap protein could specifically bind to PCV2polyclonal antibody,HRP-labeled anti-Histag mouse monoclonal antibody.A large number of regular VLPs with a diameter of about 17-20nm could be observed under the transmission electron microscope.Both vaccines could induce and stimulate mice to produce antibody response,and the VLP group induces higher level.In summary,this study achieved the soluble expression of PCV2Cap protein in Ec.oli.Target protein can be purified by improved nickel affinity chromatography,the purified protein can spontaneously assemble into virus-like particles in vitro,can stimulate antibody response after immunization,and can be used to prepare subunit vaccines and related biological products.

关 键 词：猪圆环病毒2型 CAP蛋白 可溶性表达 病毒样颗粒 

分 类 号：S852.65[农业科学—基础兽医学]