摘要
【目的】通过原核表达并纯化获得O型口蹄疫病毒(FMDV)VP1基因及其多表位基因的重组蛋白,为建立O型FMDV抗体ELISA检测试剂盒及制备动物高免血清提供技术支持。【方法】以O型FMDV VP1全基因重组质粒p MD18-T-T-VP1及串联的VP1多表位基因重组质粒p MD18-T-O-VP1为模板,通过特异性引物扩增并回收目的基因,构建重组质粒p ET-32a-VP1和p GEX-6p-1-VP1,然后转入大肠杆菌BL211(DE3)中诱导表达,并以SDS-PAGE和Western blotting对融合蛋白进行分析鉴定。【结果】O型FMDV的VP1基因及其多表位基因在大肠杆菌BL21(DE3)中得到正确表达,表达的两种融合蛋白主要以包涵体形式存在,纯度较高,且均能与猪抗O型FMDV阳性血清发生特异性结合,具有良好的反应原性。【结论】表达获得的融合蛋白具有良好的反应原性,可作为包被抗原应用于O型FMDV抗体检测试剂盒研发。
【Objective】The present experiment was conducted to obtain recombinant proteins coded by VP1 gene and multi-epitope gene of foot-and-mouth disease virus（FMDV） serotype O by prokaryotic expression and chromatographic purification, in order to provided technical supports for developing ELISA detection kit to detect antibody to FMDV serotype O and preparing high immune serum for animals. 【Method】Using recombinant plasmid p MD18T-T-VP1 containing full-length VP1 gene of FMDV serotype O and p MD18T-O-VP1 containing VP1 multi-epitope gene as templates, the target genes were amplified with specific primers, and then recycled, so as to construct expression plasmids PET-32a-VP1 and p GEX-6p-1-VP1. Then, the recombinant plasmids were transfered into Escherichia coli BL21（DE3）, and recombinant strains were induced to express target gene. Finally, the obtained fusion proteins were identified by SDS-PAGE and Western blotting analysis. 【Result 】The results showed that, the structural protein VP1 gene and multi-epitope gene of FMDV serotype O were expressed successfully in E. coli BL21（DE3）. And the two obtained fusion proteins mainly existed in the form of inclusion body, had high purity after purification, and which could specifically react with porcine antiserum to FMDV serotype O, therefore they had all strong reactionogenicity. 【 Conclusion 】 The obtained fusion proteins have strong reactionogenicity, thus which can be used as coating antigen to further develop antibody detection kit of FMDV serotype O.
出处
《南方农业学报》
CAS
CSCD
北大核心
2016年第3期472-477,共6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)
关键词
O型FMDV
VP1基因
多表位基因
原核表达
纯化
鉴定
FMDV serotype O
VP1 gene
multi-epitope gene
prokaryotic expression
purification
identification
