FMDV非结构蛋白2C主要表位区与3AB基因的组合表达与活性分析

Prokaryotic expression and bioreactivity analysis of a major epitope region of 2C with 3AB within non-structural protein of foot-and-mouth disease virus

摘要:

近年的研究表明, 口蹄疫病毒(FMDV)非结构蛋白(NSP)2C在区分灭活疫苗免疫动物与自然感染动物方面具有潜在的价值, 为了建立敏感性更高的鉴别诊断方法, 将截取了2C蛋白N-端和C-端的主要B细胞表位区和完整3AB蛋白基因组合后, 进行了原核表达, 得到了分子量约为47.6 kD的目的蛋白2C￠3AB。通过Western blotting分析证明, 表达产物能被FMDV感染动物阳性血清识别, 具有很好的反应性。以通过电泳纯化的目的蛋白作抗原进行间接ELISA检测不同来源的动物血清, 结果表明, 该抗原仅与感染动物血清具有很好的反应活性, 而与健康动物与免疫动物血清无反应, 说明重组蛋白2C￠3AB可作为区分灭活疫苗免疫动物与感染动物的鉴别诊断抗原。用2C￠3AB和3ABC为抗原进行间接ELISA, 对比检测田间血清样品, 结果显示2C￠3AB-ELISA敏感性比3ABC-ELISA高。说明以重组蛋白2C￠3AB作为鉴别诊断抗原, 能进一步提高对临界值血清的检出率, 这对区分灭活疫苗免疫动物与FMDV隐性感染动物与带毒动物具有非常重要的意义。

Abstract:

In recent years, the potential value of nonstructural protein (NSP) 2C was well documented for distinguishing foot-and-mouth disease virus (FMDV) in infected animals and vaccinated animals. In order to develop a more sensitive approach to detect natural infected FMDV while there is no interact with vaccinated FMDV, we incorporated a major epitope region of 2C with whole 3AB coding region within NSP and expressed in Escherichia coli. We got a 47.6 kD fusion protein named 2C￠3AB. The product showed a specific reactivity with FMDV from serum of infected animal by using Western blotting analysis. This suggests that this protein could be applied to distinguish infected FMDV and vaccinated FMDV. We further compared 2C￠3AB protein with 3ABC fusion protein, another available protein used for detecting infected FMDV, using indirect ELISA assay. The results showed that 2C￠3AB-ELISA had higher sensitivity than that of 3ABC-ELISA for distinguishing infected FMDV and vaccinated FMDV of sera from epidemic region. Therefore, this recombinant protein 2C￠3AB is a good candidate protein to develop more sensitive method to differentiate infected FMDV and vaccinated FMDV from vaccinated animals. This finding will increase our capability to check the infectious virus carrier and finally improve FMDV infection control.