变异猪流行性腹泻病毒M和N融合双基因的原核表达及表达产物免疫原性分析

Prokaryotic Expression and Immunogenic Analysis of the Combined M and N Fusion Genes for Variant Porcine Epidemic Diarrhea Virus

旨在构建变异猪流行性腹泻病毒(PEDV)的M和N双基因融合质粒,并分析表达蛋白免疫原性。通过扩增变异PEDV FJFQ2014毒株(GenBank登陆号KJ646580)的M基因和N基因,将M基因克隆至pEASY-Blunt E2(pE2)表达载体,构建出pE2-M质粒。利用同源重组原理,采用无缝拼接试剂盒将N基因克隆至E2-M片段,转化到Trans 1-T1感受态细胞中,构建出pE2-M-N融合双基因质粒并转化Transetta(DE3),表达出相应的融合蛋白,采用SDS-PAGE和Western blot验证蛋白的表达情况。将纯化的M-N融合蛋白皮下接种BLAB/c小鼠,通过ELISA方法检测融合蛋白的免疫原性。结果表明:扩增出M和N基因,成功构建了pE2-M质粒,并将E2-M基因与N基因进行有效连接,构建出pE2-M-N融合双基因质粒,表达出了具有免疫原性的M-N融合蛋白,该蛋白能够诱导小鼠产生相应的特异性抗体。变异PEDV的pE2-M-N双基因融合质粒的构建,为进一步开展变异PEDV的诊断技术和免疫学等研究,奠定良好基础。

The aim of this study was to construct a double gene fusion plasmid of the membrane（M）and nucleocapsid（N）genes of variant porcine epidemic diarrhea virus（PEDV）and analyze the immunogenicity of the expressed proteins.The M and N genes were amplified by PCR from PEDV variant isolate,FJFQ2014（GenBank No.KJ646580）.First,the M gene was cloned into pEASY-Blunt E2（pE2）vector to construct pE2-M plasmid.Then the amplified N gene and E2-M gene of expression vector pE2-M were cloned into the prokaryotic expression vector,resulting a recombinant plasmid pE2-M-N.The plasmid was transformed to Transetta（DE3）for protein expression.The expressed product was identified by SDS-PAGE and Western blot.The BLAB/c mice were immunized with the purified PEDV M-N protein and the antibody titer was determined by ELISA.The results indicated that PEDV M-N fusion protein was successfully expressed in Transetta（DE3）and had immunogenicity,and induced anti-PEDV antibody in mice.The pE2-M-N plasmid of variant PEDV was constructed and expressed successfully.This study lays a foundation for the development of the serodiagnostic methods and immunology research for PEDV.