PPV-VP2蛋白和PCV2-Cap蛋白二联亚单位疫苗的研究  

Duplex subunit vaccine combined with PPV VP2 and PCV2 Cap proteins

作　　者：刘国阳 华涛[2] 乔绪稳[2] 唐波[2] 常晨[2] 侯继波[2] 李锦春[1] 张道华[2] LIU Guoyang;HUA Tao;QIAO Xuwen;TANG Bo;CHANG Chen;HOU Jibo;LI Jinchun;ZHANG Daohua(College of Animal Science and Technology,Anhui Agricultural University,Hefei,Anhui 230036,China;National Research Center of Engineering and Technology for Veterinary Biologicals,Jiangsu Academy of Agricultural Sciences,Nanjing,Jiangsu 210014,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]江苏省农业科学院/国家兽用生物制品工程技术研究中心,江苏南京210014

出　　处：《西北农林科技大学学报（自然科学版）》2018年第9期18-26,34,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：公益性(农业)行业科研专项(201303046);江苏省农业科技自主创新项目[CX(15)1059]

摘　　要：【目的】研制PCV2ORF2基因编码的Cap蛋白与PPV VP2结构蛋白二联亚单位疫苗。【方法】设计截去NLS信号肽序列的Cap蛋白氨基酸序列和VP2蛋白氨基酸全序列,分别进行大肠杆菌偏嗜密码子优化,构建重组表达质粒,利用大肠杆菌表达系统表达出可溶性的Cap蛋白和VP2重组蛋白。将重组蛋白纯化后分别与ISA201佐剂混合制备单苗和二联亚单位疫苗,免疫ICR小鼠,同时设立PBS空白对照和大肠杆菌BL21对照。首免后21d进行加强免疫,利用MTT比色法、细胞中和试验法和ELISA法对免疫小鼠淋巴细胞的转化功能及细胞因子(IL-4、IL-10、IFN-γ、TNF-α)mRNA水平、免疫后中和抗体效价以及ELISA抗体水平进行检测。小鼠二免21d后进行PPV和PCV2混合攻毒,利用Real-time PCR法定量测定攻毒后小鼠脾脏的病毒含量。【结果】成功构建了重组质粒pET32a-Cap和pET32a-VP2,表达出可溶性蛋白Cap和VP2,经GEM颗粒和饱和硫酸铵沉淀法纯化蛋白,获得的PCV2Cap蛋白质量浓度为1 213μg/mL,PPV VP2蛋白质量浓度为1 025μg/mL。两种蛋白混合乳化制苗后无相互免疫抑制作用,并且能够诱导机体产生良好的体液免疫和细胞免疫应答。免疫后42d,单苗免疫组和二联亚单位疫苗组PCV2ELISA抗体效价均能达到800以上,中和抗体达到32~38,PPV HI抗体效价均高于6,PPV中和效价高于300,并且诱导机体产生强烈的淋巴细胞增殖反应,提高IL-4和IFN-γ的mRNA表达水平。攻毒试验结果显示,免疫组小鼠脾脏中PCV2和PPV含量显著低于对照组,免疫组组间无显著性差异。【结论】利用大肠杆菌表达的PCV2Cap蛋白和PPV VP2蛋白制备的二联亚单位疫苗免疫ICR小鼠,其抗体水平与Cap、VP2单苗免疫水平相当,且对ICR小鼠有较好的免疫保护作用。【Objective】This study investigated the immunogenicity of combined subunit vaccine of PCV2 Cap and PPV VP2.【Method】The sequences of Cap and VP2 with NLS truncation were designed and the PCV2 ORF2 and PPV VP2 gene codons were optimized.Then,they were cloned into expression vector pET32a and induced for expression in E.coli BL21(DE),individually.SDS-PAGE and Western blotting results confirmed that they could be expressed with the production of soluble proteins.After purification,the two proteins were mixed with ISA201 adjuvant,respectively.Mice were inoculated with VP2/Cap vaccine,VP2 vaccine and Cap vaccine,and empty vector was developed as control.The immune efficacy was determined by MTT assay,neutralization tests and ELISA,respectively.【Result】The recombinant plasmids pET32a-Cap and pET32a-VP2 were built to express the Cap and VP2 proteins.The concentrations were 1 213μg/mL and 1 025μg/mL after purification.There was no mutual antagonistic after inoculated with the subunit vaccine mixed by proteins.The vaccine induced to produce good humoral immunity and cellular immune response.After 42 days of vaccination,the single vaccine groups and Duplex subunit vaccine group PCV2 antibody titers reached more than 800,neutralizing antibodies were 32-38;and PPV HI antibodies were higher than 6,neutralizing antibodies were higher than 300.The subunit vaccine also induced the lymphocyte proliferation strongly and improved the IL-4 and IFN-γmRNA expression levels.The challenge with PCV2 and PPV showed that the virus loads in spleen in immunized groups were significantly lower than that of the control group.【Conclusion】The vaccine prepared with two proteins and ISA201 could induce immune protection in mice against PCV2 and PPV infection.

关 键 词：CAP蛋白 VP2蛋白 可溶性表达 二联亚单位疫苗 猪细小病毒 猪圆环病毒 

分 类 号：S854.4[农业科学—临床兽医学]