口蹄疫病毒非结构蛋白3AB 的原核表达及应用

Expression and utilization of 3AB nonstructural protein of foot-and-mouth disease virus in Escherichia coli

摘要:

旨在建立一种检测口蹄疫病毒非结构蛋白抗体的敏感、特异的ELISA 方法。克隆、表达了口蹄疫病毒非结构蛋白3AB 基因，原核表达的重组蛋白经亲和层析法纯化及Western blotting 鉴定后作为包被抗原，建立检测口蹄疫病毒非结构蛋白抗体的3AB 间接ELISA 方法，通过与商品化试剂盒3ABC-ELISA 的比对试验对其进行评价。结果显示，重组蛋白3AB 以包涵体形式表达；能与口蹄疫病毒感染血清发生特异性反应，而不能与疫苗免疫动物血清发生反应；在检测田间样品时，与3ABC-ELISA 具有同样的特异性

关键词:口蹄疫病毒; 蛋白; 反转录聚合酶链反应 (RT-PCR); 酶联免疫反应 (ELISA)

Abstract:

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA His·Bind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.

Key words:foot-and-mouth disease virus; protein; RT-PCR; ELISA