口蹄疫病毒非结构蛋白2C基因主要表位区的表达及活性检测

Expression of Major B-cell Epitopes within 2C Non-structural Protein of FMDV and Its Bioactivity

根据测定的口蹄疫病毒(FMDV)2C基因序列,设计了一对特异性的表达引物,用于扩增2C基因5′-端174bp和3′-端279 bp连接片段,该区含有较丰富的B细胞表位编码序列。扩增片段通过NcoⅠ和SalⅠ酶切位点插入表达载体pET-30a。序列测定表明,目的基因片段连接正确,按正确的读码框插入表达载体中。重组表达质粒转化BL21(DE3)pLys,经IPTG诱导表达目的蛋白。表达产物经SDS-PAGE和Western Blotting检测表明,2C基因主要表位区成功地在大肠杆菌表达,表达产物为约23 ku的融合蛋白,能够与FMDV感染动物血清发生特异性反应,而不与健康和灭活疫苗免疫动物血清反应。该研究为建立鉴别FMDV自然感染动物和灭活疫苗免疫动物的酶联免疫转印(EITB)方法提供了所需的材料。

A pair of primer was designed according to the 2C gene sequence of foot-and-mouth disease virus （FMDV） for amplification of a fragment including 174 bp 5＇-end and 279 bp 3＇-end of 2C gene, which encoded abundant B-cell epitopes of 2C protein. The amplified fragment was inserted into pET-30a plasmid （Novagen） via two unique restriction sites of Nco I and Sal 1 . Open reading frame of target gene was confirmed correctly inserted into the positive recombinant plasmid by sequencing, the recombinant plasmid was transformed into the host strain bacteria BL21 （DE3）pLys for protein expression. After inducing by IPTG at 37℃ for five hours, the expressed product was analyzed by SDS-PAGE and Western Blotting. The results revealed that the target gene fragment of 2C had been expressed successfully. The product is a 23 kD fusion protein and can react with sera derived from FMDV infected animal. It would provide an useful antigen for establishment of enzyme linked immune transfer blot （EITB） diagnostic method, which can be used for differentiation of the FMDV infected animals from the vaccinated animals.