特异性靶向犬细小病毒融合DNA疫苗的构建及免疫效果的研究

doi:10.16431/j.cnki.1671-7236.2016.10.038

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (10): 2768-2774.doi: 10.16431/j.cnki.1671-7236.2016.10.038

特异性靶向犬细小病毒融合DNA疫苗的构建及免疫效果的研究

吴植^1,2, 郝福星¹, 王永娟¹, 刘洋¹, 王栋¹, 徐州¹, 袁维峰³

1. 江苏农牧科技职业学院, 泰州 225300;
2. 南京农业大学动物医学院, 南京 210095;
3. 中国农业科学院北京畜牧兽医研究所, 北京 100193

收稿日期:2016-03-30 出版日期:2016-10-20 发布日期:2016-10-28
通讯作者: 袁维峰 E-mail:yuanweifeng@126.com
作者简介:吴植(1980-),男,江苏东台人,硕士,研究方向:兽医微生物与免疫学,E-mail:tzwuzhi@163.com
基金资助:
江苏农牧科技职业学院院级课题（NSFYB1302）；公益性行业（农业）科研专项经费项目（201303042）；中国农业科学院科技创新工程（ASTIP-IAS-11）

Construction of Specifically Targeted Chimeric Canine Parvovirus DNA Vaccine and Its Efficacy Assessment

WU Zhi^1,2, HAO Fu-xing¹, WANG Yong-juan¹, LIU Yang¹, WANG Dong¹, XU Zhou¹, YUAN Wei-feng³

1. Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China;
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
3. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2016-03-30 Online:2016-10-20 Published:2016-10-28

摘要/Abstract

摘要：

研究旨在构建特异性靶向犬细小病毒（canine parvovirus，CPV）融合DNA疫苗，探讨其诱导机体产生免疫应答的效果。试验用重组技术构建了含细胞毒性T淋巴细胞抗原-4胞外区（CTLA-4₁₂₅）与犬细小病毒VP2的主要抗原表位区域（VP2₂₂₈）融合表达质粒pVAX1-CTLA-4₁₂₅-VP2₂₂₈，同时构建了不含CTLA-4₁₂₅的pVAX1-VP2₂₂₈，体外转染COS-7细胞，Western blotting检测其表达产物；将pVAX1-CTLA-4₁₂₅-VP2₂₂₈、pVAX1-VP2₂₂₈分别免疫小鼠，免疫后进行抗体水平测定和抗体亚型分析；通过淋巴细胞增殖试验和ELISA分别检测淋巴细胞刺激指数和γ-干扰素表达水平。结果显示成功构建了pVAX1-CTLA-4₁₂₅-VP2₂₂₈和pVAX1-VP2₂₂₈，并能在COS-7细胞中正确表达；抗体检测结果显示pVAX1-CTLA-4₁₂₅-VP2₂₂₈免疫组抗体水平显著高于pVAX1-VP2₂₂₈免疫组（P<0.05）；淋巴细胞增殖试验显示pVAX1-CTLA-4₁₂₅-VP2₂₂₈免疫组的刺激指数显著高于pVAX1-VP2₂₂₈免疫组（P<0.05）；γ-干扰素表达水平测定显示pVAX1-CTLA-4₁₂₅-VP2₂₂₈免疫组极显著高于pVAX1-VP2₂₂₈免疫组（P<0.01）。结果表明，CTLA-4₁₂₅-VP2₂₂₈融合DNA能有效增强动物对VP2₂₂₈抗原的免疫应答，为进一步研究融合DNA疫苗特异性的靶向递呈机制奠定基础。

关键词: 犬细小病毒; DNA疫苗; 抗原递呈细胞; 靶向性

Abstract:

To design chimeric DNA vaccine targeted to antigen-presenting cells with enhanced efficacy to induce immunization.The plasmid containing the gene encoding the extracelluar domain of canine cytotoxic T-lymphocyte antigen 4(CTLA-4₁₂₅),was directly fused to the gene fragment for major antigenic epitopes of VP2(VP2₂₂₈) by molecular engineering technology.The plasmid containing VP2₂₂₈ alone was also constructed to serve as control and transfection of COS-7 cells with the two resultant plasimds was performed respectively,followed by assay of Western blotting.The mice were immunized with the constructed two plasimds,respectively.After immunization,the antibodies anginst CPV in the immunized mice at different times were measured by HI.The spleen lymphocyte proliferation response was determined by lymphocyte proliferation assay,and the interferon-γ (IFN-γ) expression level of the mouse lymphocytes were measured by ELISA.The eukaryotic expression plasimds of CTLA-4₁₂₅-VP2₂₂₈ fusion protein or VP2₂₂₈ alone were cloned.Western blotting showed that the two recombinant proteins could be expressed.Immunization results showed that the antibody levels in serum of CTLA-4₁₂₅-VP2₂₂₈-immunized mice were significantly higher than that of VP2₂₂₈-immunized mice (P<0.05).The lymphocyte stimulation indexes and secreted IFN-γ levels of the CTLA-4₁₂₅-VP2₂₂₈-immunized mice were significantly higher than that of VP2₂₂₈-immunized mice (P<0.05 and P<0.01),respectively.CTLA-4₁₂₅-VP2₂₂₈ chimeric DNA vaccine stimulates strong immune response in mice,making it possible for further exploration into chimeric DNA that target the antigen to APCs.

Key words: canine parvovirus; DNA vaccine; antigen-presenting cells; targeting

中图分类号:

R392

吴植, 郝福星, 王永娟, 刘洋, 王栋, 徐州, 袁维峰. 特异性靶向犬细小病毒融合DNA疫苗的构建及免疫效果的研究[J]. 《中国畜牧兽医》, 2016, 43(10): 2768-2774.

WU Zhi, HAO Fu-xing, WANG Yong-juan, LIU Yang, WANG Dong, XU Zhou, YUAN Wei-feng. Construction of Specifically Targeted Chimeric Canine Parvovirus DNA Vaccine and Its Efficacy Assessment[J]. , 2016, 43(10): 2768-2774.

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特异性靶向犬细小病毒融合DNA疫苗的构建及免疫效果的研究

Construction of Specifically Targeted Chimeric Canine Parvovirus DNA Vaccine and Its Efficacy Assessment

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