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

doi:10.11843/j.issn.0366-6964.2018.06.017

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (6): 1249-1255.doi: 10.11843/j.issn.0366-6964.2018.06.017

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

王隆柏, 王晨燕, 吴学敏, 陈秋勇, 车勇良, 陈如敬, 周伦江*

福建省农业科学院畜牧兽医研究所/福建省畜禽疫病防治工程技术研究中心, 福州 350013

收稿日期:2017-11-14 出版日期:2018-06-23 发布日期:2018-06-23
通讯作者: 周伦江,E-mail:lunjiang@163.com
作者简介:王隆柏(1977-),男,福建宁化人,副研究员,硕士,主要从事分子生物学及免疫学研究,Tell:0591-87572301,E-mail:wanglongbai@163.com
基金资助:
公益类科研院所专项（2018R1023-4）；福建省自然科学基金项目（2015J01112）；公益类科研院所专项（2017R1023-8）

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

WANG Long-bai, WANG Chen-yan, WU Xue-min, CHEN Qiu-yong, CHE Yong-liang, CHEN Ru-jing, ZHOU Lun-jiang*

Institute of Animal Husbandry and Veterinary Medicine/Agricultural Industrialization Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China

Received:2017-11-14 Online:2018-06-23 Published:2018-06-23

摘要/Abstract

摘要：

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。

Abstract:

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.

中图分类号:

王隆柏, 王晨燕, 吴学敏, 陈秋勇, 车勇良, 陈如敬, 周伦江. 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[J]. 畜牧兽医学报, 2018, 49(6): 1249-1255.

WANG Long-bai, WANG Chen-yan, WU Xue-min, CHEN Qiu-yong, CHE Yong-liang, CHEN Ru-jing, ZHOU Lun-jiang. Prokaryotic Expression and Immunogenic Analysis of the Combined M and N Fusion Genes for Variant Porcine Epidemic Diarrhea Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1249-1255.

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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

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