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

Chinese. 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。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。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。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。

English. The study was conducted to express nonstructural protein VPg1-2, VPg2-3, VPg3 of the foot-and-mouth disease virus ( FMDV ) for differentiation of the infected and vaccinated animal VPg1-2 (3B1-2),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 VPg1-2 protein as antigen, and 4 groups of background-known cattle sera were tested. The expressed VPg1-2, 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 VPg1-2 lasted for at least 90 ays. The earliest appearing time of VPg1-2 antibody was10 days after infection. The expressed VPg1-2 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.