变异猪流行性腹泻病毒M和N双基因融合真核表达及其活性鉴定

Eukaryotic expression and active identification of the M and N double genes fusion plasmid for variant porcine epidemic diarrhea virus

【目的】体外表达变异猪流行性腹泻病毒(PEDV)的M-N双基因融合蛋白并明确其生物活性,为开展PEDV新型疫苗及其诊断技术研究提供更多的候选蛋白。【方法】将变异PEDV的M基因克隆至pEASY-Blunt M2(pM2)真核表达载体构建pM2-M真核质粒,通过同源重组将N基因克隆至M2-M基因片段构建pM2-M-N双基因融合重组质粒,然后转染293T细胞表达出M-N双基因融合蛋白。通过Western blotting、ELISA及免疫BLAB/c小鼠试验鉴定表达融合蛋白的生物学活性。【结果】扩增获得的M基因、N基因片段大小分别为678和1359 bp,且与原序列的相似性均达100.0%。将M基因和N基因并连接至pM2真核表达载体成功构建获得pM2-M-N双基因融合重组质粒,M-N双基因片段大小为2004 bp,且与原序列的相似性为99.3%。以pM2-M-N双基因融合重组质粒转染239T细胞,表达出大小约75 kD且具有免疫活性的M-N双基因融合蛋白,纯化的M-N双基因融合蛋白能与PEDV阳性血清反应,与猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)等病毒均无交叉反应;以其免疫BLAB/c小鼠能诱导产生特异性抗体。【结论】通过pM2真核表达载体能构建获得变异PEDV毒株的pM2-M-N融合双基因重组质粒,转染293T细胞后可表达出具有良好免疫活性的M-N双基因融合蛋白,为后期开展变异PEDV基因工程疫苗及其诊断技术研究提供更多的候选蛋白。

【Objective】The protein of M-N double gene fusion protein plasmid of variant porcine epidemic diarrhea virus(PEDV)was expressed in vitro,and clarified the immunological activity of the protein,more candidate fusion proteins were provided to carry out research on new vaccines and diagnostic technologies for PEDV.【Method】The M gene of variant PEDV was cloned into pEASY-Blunt M2(pM2)eukaryotic expression vector and pM2-M eukaryotic plasmid was built,then N gene was cloned into M2-M gene fragment to construct pM2-M-N double gene fusion recombinant plasmid by homologous recombination,and transformed into 293T cell,M-N double gene fusion protein was expressed accordingly.Biological activity of expressing fusion protein was tested by Western blotting,ELISA and immunity BLAB/c mice test.【Result】The amplified fragments of M gene and N gene were 678 and 1359 bp respectively,and the similarities compared with the original sequence were both 100.0%.The M gene and N gene were linked into pM2 eukaryotic expression vector,and pM2-M-N double gene fusion recombinant plasmid was built.The amplified fragments of M-N double fusion genes was 2004 bp,and the similarity was compared with the original sequence was 99.3%.pM2-M-N double gene fusion recombinant plasmid transfected 239T cells,and M-N double gene fusion protein(about 75 kD)with immunological competence.The purified M-N double gene fusion protein could react with PEDV positive serum.The fusion protein had no crossreactions with porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),porcine circovirus type 2(PCV2),classical swine fever virus(CSFV)and porcine transmissible gastroenteritis virus(TGEV).The BLAB/c mice immunized by it could produce specific antibody.【Conclusion】The pM2-M-N fusion double gene recombinant plasmid of variant PEDV can be constructed by pM2 eukaryotic expression vector,the M-N double fusion protein with immunological competence is expressed after 293T cell being transfected.More candidate fusion proteins are provided to carry out genetic engineering vaccines and diagnostic techniques for variant PEDV.