H5N1亚型禽流感病毒核蛋白的原核表达与鉴定  

Prokaryotic expression and identification of a nucleoprotein of the H5N1 subtype avian influenza virus

作　　者：祁宇 窦悦 陈明涛 张亚敏 赵尧烨 杨争艳 王铁成 赵永坤 李元果 孙伟洋 冯娜 任志广 高玉伟 夏咸柱 QI Yu;DOU Yue;CHEN Ming-tao;ZHANG Ya-min;ZHAO Yao-ye;YANG Zheng-yan;WANG Tie-cheng;ZHAO Yong-kun;LI Yuan-guo;SUN Wei-yang;FENG Na;REN Zhi-guang;GAO Yu-wei;XIA Xian-zhu(Joint National Laboratory for Antibody Drug Engineering,Henan University,School of Basic Medical Sciences,Kaifeng,China 475004;Military Veterinary Institute,Academy of Military Medical Sciences)

机构地区：[1]河南大学基础医学院,抗体药物开发技术国家地方联合工程实验室,河南开封475004 [2]军事科学院军事医学研究院军事兽医研究所

出　　处：《中国病原生物学杂志》2020年第5期508-511,517,共5页Journal of Pathogen Biology

基　　金：国家重点研发计划项目(No.2017YFD0501705);国家自然科学基金项目(No.31902287,81803575);河南省重点研发与推广专项(科技攻关)计划项目(No.192102310301);开封市重点研发与推广专项(科技攻关)计划项目(No.1903018)。

摘　　要：目的原核表达系统表达H5N1亚型禽流感病毒核蛋白(nuclear protein,NP)。方法从H5N1A/meerkat/Shanghai/2012(SH-1),2.3.2.1中提取总RNA,反转录为cDNA后采用PCR扩增NP基因,构建重组质粒经双酶切、测序鉴定正确后转入BL21(DE3)感受态细胞,IPTG诱导重组蛋白的表达,纯化后进行结合活性鉴定。结果构建的重组质粒H5N1NP经双酶切及测序鉴定正确,转化入大肠埃希菌后表达NP蛋白,SDS-PAGE分析其相对分子质量为70×103,与理论值相符。ELISA法检测NP蛋白与相应抗体具有结合活性。结论实现了禽流感病毒H5N1NP重组蛋白的原核表达,表达蛋白具有结合活性,为进一步研究NP蛋白的功能和诊断用单抗的研发奠定了基础。Objective To express a nuclear protein(NP)of the H5 N1 subtype avian influenza virus in a prokaryotic expression system.Methods Total RNA was extracted from H5 N1 A/meerkat/Shanghai 2012(SH-1),2.3.2.1,and then the NP gene was amplified using PCR.The recombinant plasmid was constructed and transformed into competent BL21(DE3)cells via double enzyme digestion and sequencing.Expression of the recombinant protein was induced with IPTG;after purification,its binding activity was determined.Results The recombinant plasmid H5 N1 NP was confirmed as correct using double restriction enzyme digestion and sequencing.The recombinant plasmid was transformed into E.coli and NP protein was expressed.SDS-PAGE indicated that the relative molecular weight of the recombinant plasmid was 70×103,which was consistent with the theoretical value.ELISA indicated that the NP protein had binding activity with the corresponding antibody.Conclusion A recombinant H5 N1 avian influenza NP was successfully expressed in E.coli,and the expressed protein has binding activity.This finding lays the foundation for further research on the function of the NP protein and the development of a monoclonal antibody for diagnostic use.

关 键 词：H5N1流感病毒 NP蛋白 原核表达 

分 类 号：R37[医药卫生—病原生物学]