基于SpyTag/SpyCatcher系统的猪细小病毒-猪圆环病毒2型重组蛋白疫苗的制备及其免疫原性评价  

Preparation and immunogenicity evaluation of PPV-PCV2 recombinant protein vaccine based on SpyTag/SpyCatcher system

作　　者：许咏雯 冯金 陈丽云 彭涛 许煜华 陈忠正[1] XU Yongwen;FENG Jin;CHEN Liyun;PENG Tao;XU Yuhua;CHEN Zhongzheng(Collge of Food Science,South China Agricultural University,Guangzhou 510640,Guangdong Province,China;不详)

机构地区：[1]华南农业大学食品学院,广东广州510640 [2]广东华南疫苗股份有限公司,广东广州510700

出　　处：《中国生物制品学杂志》2024年第11期1286-1293,共8页Chinese Journal of Biologicals

基　　金：广州市产业领军人才聚集工程项目(CYLJTD-201602);广东省重点领域研发计划项目(2020B111109003)。

摘　　要：目的基于SpyTag/SpyCatcher系统制备猪细小病毒(porcine parvovirus,PPV)-猪圆环病毒2型(porcine circovirus type 2,PCV2)二联亚单位疫苗,并评价其免疫原性,为1针防治PPV与PCV2混合感染的候选二联亚单位疫苗的研发奠定基础。方法以猪细小病毒1型(porcine parvovirus type 1,PPV1)VP2蛋白为骨架,将SpyTag肽段插入VP2蛋白的loop2区域,利用昆虫细胞杆状病毒系统表达该融合蛋白并记为PPV-ST。选取PCV2 Cap蛋白的5个B细胞表位、Rep蛋白的3个T细胞抗原表位,将其串联,并在N-端融合SpyCatcher肽段,利用原核表达系统表达并记为SCPCV2。将纯化蛋白PPV-ST与SC-PCV2按摩尔比1∶1混合,25℃孵育6 h共价组装,以ISA 201矿物油为佐剂制备疫苗。按低、中、高(1、10、20μg/只)剂量肌内注射免疫雌性BALB/c小鼠,14 d后进行加强免疫。采用间接ELISA法检测免疫小鼠血清中特异性IgG抗体水平,ELISpot法检测小鼠脾淋巴细胞分泌的相关细胞因子IL-4和IFNγ水平。结果重组杆状病毒构建成功且能表达重组蛋白PPV-ST,原核系统也成功表达重组蛋白SC-PCV2,二者共价组装物可被鼠抗His标签抗体特异性识别,表明两个蛋白能共价组装。PPV-PCV2中、高剂量组免疫小鼠血清特异性IgG抗体效价均显著高于PBS组(P均<0.05),IL-4水平也显著高于PBS组(P均<0.05)。结论基于SpyTag/SpyCatcher系统制备的PPV-PCV2二联亚单位疫苗具有较好的免疫原性。Objective To prepare porcine parvovirus(PPV)-porcine circovirus type 2(PCV2)duplex subunit vaccine based on SpyTag/SpyCatcher system and evaluate its immunogenicity,so as to lay a foundation for the development of candidate duplex subunit vaccine for prevention and treatment of PPV and PCV2 mixed infection.Methods The VP2 protein of porcine parvovirus type 1(PPV1)was used as the backbone,the SpyTag peptide was inserted into the loop2 region of the VP2 protein,and the fusion protein was expressed using an insect cell baculovirus system and labeled as PPV-ST.Five Bcell epitopes of the Cap protein and three T-cell antigenic epitopes of the Rep protein of PCV2 were selected and tandem linked,and a SpyCatcher peptide was fused to the N-terminal end of the fusion protein.The fusion protein was expressed using a prokaryotic expression system and recorded as SC-PCV2.PPV-ST protein and SC-PCV2 protein were purified and mixed at a molar ratio of 1∶1,and the vaccine was prepared by incubation at 25℃for 6 h combined with covalent assembly,using ISA 201 mineral oil as an adjuvant.The vaccine was injected intramuscularly into female BALB/c mice at 1,10,and20μg/mouse(low,medium,and high doses),and a single vaccine group and a PBS control group were set up at the same time,and booster immunization was carried out 14 d later.Indirect ELISA was used to detect the specific IgG antibody potency in the serum of immunized mice,and ELISpot was used to detect the levels of cytokines(IL-4 and IFNγ)secreted by mouse splenic lymphocytes.Results The recombinant baculovirus was successfully constructed and could express recombinant protein PPV-ST,and the prokaryotic system also expressed recombinant protein SC-PCV2 successfully.The covalently assembled products were specifically recognized by mouse anti-His tag antibody,indicating that the two proteins could be covalently assembled.The specific IgG antibody potency in the serum of mice in both PPV-PCV2 medium-dose and highdose groups was significantly higher than that in the PBS group(

关 键 词：猪细小病毒 猪圆环病毒 重组蛋白疫苗 免疫原性 SpyTag/SpyCatcher系统 

分 类 号：S851.35[农业科学—预防兽医学]