中国农业科学 ›› 2007, Vol. 40 ›› Issue (3): 601-607 .

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

口蹄疫病毒非结构蛋白VPg1-2、VPg2-3、VPg3基因的克隆、表达及VPg1-2抗体持续时间的研究

孙 明,王宏伟,田克恭,王传彬,陈西钊,遇秀玲,金 萍,曹 振,许燕辉,赵心力   

  1. 农业部兽医诊断中心
  • 收稿日期:2005-08-29 修回日期:1900-01-01 出版日期:2007-03-10 发布日期:2007-03-10
  • 通讯作者: 王宏伟

Research on cloning and expression of VPg1-2, VPg2-3, VPg3 gene fragments of foot-and-mouth disease virus and antibody dynamics of expressed VPg1-2 protein

  1. 农业部兽医诊断中心
  • Received:2005-08-29 Revised:1900-01-01 Online:2007-03-10 Published:2007-03-10

摘要: 【目的】表达口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)非结构蛋白VPg1-2、VPg2-3、VPg3,用于FMDV感染与灭活疫苗动物鉴别诊断和其蛋白功能研究。【方法】对O 型口蹄疫病毒太保毒株3ABC 基因片段中的 VPg1-2 (3B1-2)、VPg2-3 (3B2-3)、VPg3 (3B3)基因进行扩增和亚克隆,并将它们分别插入pGEX-4T-1 表达载体构建了重组表达质粒,经IPTG诱导后,进行Western blotting 分析。以纯化的VPg1-2表达蛋白为抗原建立间接ELISA,对背景清楚的实验牛血清进行检测。【结果】表达的VPg1-2、VPg2-3蛋白可与FMDV阳性血清发生特异性反应,表达的VPg3蛋白没有反应。VPg1-2表达蛋白与对照组(C组)和灭活疫苗反复免疫组(CI组)牛血清均不发生反应,与未免疫直接攻毒组(D组)血清均发生反应,与部分免疫后攻毒组(I组)(4/7)血清发生反应,与部分I组(3/7)血清不发生反应;D组和I组VPg1-2表达蛋白抗体持续时间最长可达90 d以上,最早检出相应抗体的时间为攻毒后第10天。【结论】表达的VPg1-2抗原用于健康牛群、免疫后未感染牛群以及未免疫感染牛群的鉴别诊断的特异性、敏感性达到100%,对免疫后感染牛群进行检测时可能有一定的漏检现象。

关键词: 口蹄疫病毒, 非结构蛋白, 抗体消长

Abstract: Abstract VPg1-2, VPg2-3, VPg3 gene fragments were successfully subcloned from 3ABC gene of the foot-and-mouth disease virus ( FMDV ) Taibao strain and their expressing plasmids were constructed by inserting the target gene fragments into pGEX-4T-1 vector. The expressed proteins were analysed by Western blotting. The results showed that the expressed VPg1-2, VPg2-3 proteins reacted with FMDV positive serum while the reaction of the VPg3 protein was negative. An indirect ELISA was set up using purified VPg1-2 protein as antigen, and 4 groups of background-known cattle sera were tested. Negative results were obtained for the non-vaccinated control group and the vaccinated group and a part of the infected post-vaccination group(3/7). On the contrary, positive results were achieved for the infected group and a part of the infected post-vaccination group(4/7). In the infected group and infected post-vaccination group, the longest duration of the antibody against VPg1-2 lasted for at least 90 days. The earliest appearing time of VPg1-2 antibody was10 days after infection.

Key words: Foot-and-mouth disease virus, Non-structural protein, Antibody dynamics