畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (9): 1488-1496.doi: 10.11843/j.issn.0366-6964.2014.09.015

• 预防兽医 • 上一篇    下一篇

伪狂犬病病毒gE基因DNA疫苗在动物体内诱导的免疫应答及IL-2对其作用影响的研究

王洪光1,郝飞1,汤德元1*,张元鑫2,马萍2,罗险峰2,曾智勇1,刘霞2,刘建1,李达1   

  1. (1.贵州大学动物科学学院,贵阳 550025; 2.贵州省动物疫病预防控制中心,贵阳 550008)
  • 收稿日期:2014-01-27 出版日期:2014-09-23 发布日期:2014-09-23
  • 通讯作者: 汤德元(1964-),男,教授,博士,硕士生导师,主要从事动物传染病病原分子生物学和中西兽医结合的教学科研工作,E-mail:tdyuan@163.com
  • 作者简介:王洪光(1989-),女,安徽人,硕士生,主要从事动物传染病病原分子病毒学研究
  • 基金资助:

    贵州省科学技术基金项目(黔科合J字[2013]2110号);贵州省2014年农业攻关项目[黔科合NY(2014)3055号]

Immune Responses of Pseudorabies Virus gE DNA Vaccine in Animals and the Immune Effects of Interleukin-2 Gene on It

WANG Hong-guang1,HAO Fei1,TANG De-yuan1*,ZHANG Yuan-xin2,MA Ping2,LUO Xian-feng2,ZENG Zhi-yong1,LIU Xia2,LIU Jian1,LI Da1   

  1. (1.College of Animal Science,Guizhou University,Guiyang 550025,China;2.Animal Disease Prevention and Control Center in Guizhou Province,Guiyang 550008,China)
  • Received:2014-01-27 Online:2014-09-23 Published:2014-09-23

摘要:

为探讨伪狂犬病病毒(PRV)gE蛋白DNA疫苗在动物体内诱导免疫应答的能力以及猪白细胞介素-2对其免疫作用的影响,构建pcDNA3.1(+)-gE1和pcDNA3.1(+)-gE2-IL-2重组真核表达质粒,将其转染Vero细胞后,采用间接免疫荧光、RT-PCR和Western blotting技术检测gE基因和IL-2基因在Vero细胞中的表达;并将构建的重组质粒、空载体、灭活病毒对照及生理盐水对照分别免疫小鼠和仔猪,通过间接ELISA方法和流式细胞技术监测血清中特异性抗体及T淋巴细胞亚群含量进行免疫学评价。结果表明,pcDNA3.1(+)-gE1和pcDNA3.1(+)-gE2-IL-2重组质粒在Vero细胞中获得较高水平的表达,且表达蛋白具有较好的反应原性。重组质粒可明显提高动物机体的体液免疫应答和细胞免疫应答水平,IL-2对PRV gE DNA疫苗的免疫效果存在明显的增强作用。动物攻毒试验表明,在PRV强毒攻击下融合表达质粒和混合质粒免疫可以给小鼠提供80%以上的保护,可给免疫仔猪提供60%以上的保护,表明融合蛋白和混合蛋白对PRV的攻击具有一定的免疫保护作用。

Abstract:

To evaluate immune responses of gE DNA vaccine of pseudorabies virus (PRV) and the enhancement potency of porcine interleukin-2 for its immunogenicity,the recombinant plasmid of pcDNA3.1(+)-gE1 and pcDNA3.1(+)-gE2-IL-2 were constructed and transfected into Vero cells.The expression of gE and IL-2 gene were identified by indirect immunofluorescent assay,RT-PCR and Western blotting.The results showed that recombinant plasmids were transiently expressed in Vero cells and the protein expressed in Vero cells had good reactogenicity.Furthermore,animals were immunized by recombinant plasmids,empty vector,inactivated virus and normal saline.Indirect ELISA and determination of T lymphocyte subsets showed that the recombinant plasmids were able to induce efficient immune responses in inoculated animals.The IL-2 gene showed the significant enhancement effects on the immunogenecity of PRV gE DNA vaccine.The animals immunized with the pcDNA3.1(+)-gE2-IL-2 recombinant plasmid and pcDNA3.1(+)-gE1+ pcDNA3.1(+)-IL-2 co-immunize provided over 80% protection in mice and over 60% protection in piglets against wild type virus challenge.These results confirm that the fusion protein and hybrid protein enhances the protection against PRV through both humoral and cell-mediated immunity.

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