A型口蹄疫病毒结构蛋白VP1的原核表达、纯化及鉴定  

Prokaryotic expression, purification and identification of structural protein VP1 of foot-and-mouth disease virus serotype A

作　　者：颜健华[1] 何奇松[1] 蒋家霞[1] 冯淑萍[1] 黄胜斌[1] 韦达有 易春华 许瑞胜 梁晟[1] 熊毅[1] 

机构地区：[1]广西动物疫病预防控制中心,南宁530001 [2]桂林市动物疫病预防控制中心,广西桂林541002 [3]崇左市动物疫病预防控制中心,广西崇左532200

出　　处：《南方农业学报》2016年第2期301-305,共5页Journal of Southern Agriculture

基　　金：广西科学研究与技术开发计划项目(桂科攻0779001)

摘　　要：【目的】通过原核表达及纯化获得A型口蹄疫病毒(FMDV)结构蛋白VP1,为建立A型FMDV的ELISA诊断方法及开发安全、高效、广谱的新型基因工程疫苗提供技术支持。【方法】以含A型FMDV VP1基因的重组质粒p MD18-T-A-VP1为模板,通过特异性引物扩增A型FMDV的VP1基因,构建表达质粒p ET-32a-VP1和p GEX-6p-1-VP1,然后转入感受态细胞E.coli BL21(DE3)中诱导表达融合蛋白。【结果】诱导表达获得的VP1融合蛋白主要以包涵体形式存在,分别经His·Bind和GST·Bind柱层析纯化,SDS-PAGE分析结果表明融合蛋白纯度较高;Western blotting检测分析发现,VP1融合蛋白能与豚鼠抗A型FMDV阳性血清发生特异性结合,但不与豚鼠抗O型和Asia1型FMDV阳性血清反应。【结论】经原核表达及纯化获得的A型FMDV VP1融合蛋白具有良好的特异性和抗原性,可用于易感动物的免疫及血清抗体筛查。【Objective】The present experiment was conducted to obtain VP1 protein of foot-and-mouth disease virus（FMDV） serotype A through prokaryotic expression and chromatographic purification, in order to provide technical support for establishing ELISA method to detect FMDV serotype A and developing a safe, efficient and broad-spectrum genetic engineering vaccine. 【Method】Using recombinant plasmid p MD18T-A-VP1 as template, the VP1 gene was amplified from FMDV serotype A with specific primers. And the expression plasmid PET-32a-VP1 and p GEX-6p-1-VP1 were constructed and transfered into Escherichia coli BL21, then E. coli BL21 was induced to express fusion protein. 【Result】The results showed that, the obtained fusion protein VP1 existed mainly in the form of inclusion body. And the SDS-PAGE results showed that the fusion proteins had a high purity through His·Bind and GST·Bind column chromatography purification. Furthermore, Western blotting analysis indicated that the purified fusion protein VP1 could specifically react with antiserum of guinea pig to FMDV serotype A, but not with antiserum of guinea pig FMDV serotype O and Asia1. 【Conclusion】Based on the experimental results,it can be inferred that the fusion proteins obtained through prokaryotic expression and chromatographic purification had good specificity and antigenicity, so it can be used to detect and screen immunity and serum antibody of susceptible animal.

关 键 词：A型FMDV VP1蛋白 原核表达 纯化 鉴定 

分 类 号：S852.659.6[农业科学—基础兽医学]