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

Research on Cloning and Expression of VPg1-2,VPg2-3,VPg3 Gene Fragments of Foot-and-Mouth Disease Virus and the Antibody Dynamics of Expressed VPg1-2 Protein

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。

[Objective] The study was conducted to express nonstructural protein VPgl-2, VPg2-3, VPg3 of the foot-and-mouth disease virus （ FMDV ） for differentiation of the infected and vaccinated animal [Method] VPgl-2 （3B 1-2）,VPg2-3 （3B2-3）,VPg3 （3B3） gene fragments were successfully amplified by PCR and subcloned from 3ABC gene of the foot-and-mouth disease virus （FMDV） Taibao strain. Their expressing plasmids were constructed by inserting the target gene fragments into pGEX-4T-1 vector. The expressed proteins were analyzed by Western blotting. An indirect ELISA was set up using purified VPgl-2 protein as antigen, and 4 groups of background-known cattle sera were tested. [Results] The expressed VPgl-2, VPg2-3 proteins reacted with FMDV positive serum while the reaction of the VPg3 protein was negative. 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 VPgl-2 lasted for at least 90 days. The earliest appearing time of VPg 1-2 antibody was 10 days after infection. [ Conclusion ] The expressed VPg 1-2 proteins＇s specificity and sensitivity were 100% for differentiating infected from vaccinated in the non-vaccinated and the vaccinated and the infected cattle. May be it＇s lower level of sensitivity for the infected post-vaccination cattle.