四川农业大学学报 ›› 2011, Vol. 29 ›› Issue (04): 549-554.doi: 10.3969/j.issn.1000-2650.2011.04.021

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ILTV Glycoprotein B(gB)与鸡Interleukin-18(IL-18)真核双表达载体的构建及表达

王淑娟1, 商艳红1, 陈红英1, 邵攀峰1,2, 刘金朋1, 朱前磊1, 王子馨1, 崔保安1   

  1. 1. 河南农业大学牧医工程学院, 郑州 450002;
    2. 杭州荐量兽用生物制品有限公司, 杭州 310018
  • 收稿日期:2011-06-20 出版日期:2011-12-31 发布日期:2017-04-12
  • 通讯作者: 崔保安,教授,博导,主要从事分子病原学及分子免疫学研究,E-mail:baoancui@henau.edu.cn。 E-mail:baoancui@henau.edu.cn
  • 作者简介:王淑娟,硕士,主要从事分子免疫学和分子病毒学研究,E-mail:snowangel517@163.com。
  • 基金资助:
    国家"十一五"科技支撑计划专项(2006BAD06A08)

Construction and Expression of Eukaryotic Coexpression Vector of ILTV Glycoprotein B and Chicken Interleukin-18 Gene

WANG Shu-juan1, SHANG Yan-hong1, CHEN Hong-ying1, SHAO Pan-feng1,2, LIU Jin-peng1, ZHU Qian-lei1, WANG Zi-xin1, CUI Bao-an1   

  1. 1. College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China;
    2. Hangzhou Jian Liang Veterinary Biological Preparations Co. LTD, Hangzhou 310018, China
  • Received:2011-06-20 Online:2011-12-31 Published:2017-04-12

摘要: 将ILTV gB基因和鸡IL-18基因插入到真核双表达载体pIRES中,构建共表达gBIL-18基因的重组质粒pIRES-gB/IL18和表达gB基因的pIRES-gB质粒。通过脂质体将其转染鸡胚成纤维细胞,利用RT-PCR及间接免疫荧光检测重组质粒在体外的表达。将重组质粒通过腿部肌肉多点注射免疫21日龄雏鸡,应用ELISA和流式细胞仪分别检测免疫鸡的体液免疫及细胞免疫水平。结果显示,pIRES-gB/IL18和pIRES-gB转染细胞中分别含gB/IL-18基因的mRNA和gB基因的mRNA。间接免疫荧光检测表明,pIRES-gB/IL18和pIRES-gB在CEF细胞中有效地转录并表达gB蛋白。pIRES-gB/IL18和pIRES-gB免疫雏鸡后均能促进外周血T淋巴细胞亚群数量和血清中特异性抗体水平的增加,但前者的免疫效果明显优于后者,表明gB基因和鸡IL-18基因均获得了表达,且鸡IL-18能明显增强ILTV DNA疫苗诱发的免疫应答。

关键词: 传染性喉气管炎病毒(ILTV), glycoprotein B(gB), 鸡Interleukin-18基因, 真核表达, DNA疫苗

Abstract: The research was to construct eukaryotic coexpression vector of avian infectious laryngotracheitis virus(ILTV) gB and chicken IL-18, and its immunogenicity was evaluated.The gB gene of ILTV and chicken IL-18 gene were inserted into the bicistronic plasmid pIRES to construct recombinant plasmids pIRES-gB and pIRES-gB/IL18.Plasmids were transfected into the chicken embryo fibroblasts(CEF) cells by using lipofectamine, respectively, and the mRNA was detected by RT-PCR and the expressed products were detected by indirect immunofluorescence assay.The 21-day-old chickens were immunized intramuscularly with plasmids, and the induced humoral and cellular responses were evaluated.gB mRNA expression was found in CEF cells and gB protein expression in CEF cells was detected by the indirect immunofluorescence test.Immunization with pIRES-gB/IL18 led to a more dramatic augmentation of humoral and T cell responses than pIRES-gB which demonstrated that ILTV gB and cIL-18 gene were expressed, and IL-18 could significantly enhance the immune response of ILTV DNA vaccine.

Key words: infectious laryngotracheitis virus, gB gene, chicken IL-18 gene, eukaryotic expression, DNA vaccine

中图分类号: 

  • S852.65