H9N2亚型禽流感病毒NA和M2基因的克隆与表达
|
摘要
旨在分别构建NA蛋白和M2蛋白的重组质粒,并进行表达鉴定。采用PCR扩增了H9N2分离株A/chicken/Shandong/LY1/2017毒株的NA基因和M2基因。同源性分析证实,这2个基因序列与目前流行毒株的基因同源性很高。将NA基因克隆至pcold1原核表达载体,将M2基因克隆至pET28a原核表达载体,成功构建了pcold1-NA和pET28a-M2重组质粒,并成功表达出相应的融合蛋白。然后对表达的融合蛋白进行纯化,并采用免疫印迹验证其免疫反应性。本试验为进一步研究H9N2亚型禽流感病毒致病机制及检测技术奠定了基础。
The purpose of this study was to construct recombinant plasmids of NA and M2 proteins and identify their expression. The NA and M2 genes of the H9N2 strains A/chicken/Shandong/LY1/2017 were amplified by PCR, respectively. Homology analysis was performed and the result confirmed that the two gene sequences had high homology with that of the current epidemic H9N2 strains. The NA gene was cloned into the prokaryotic expression vector pcold1 and the M2 gene was cloned into the prokaryotic expression vector pET28a. Recombinant plasmids of pcold1-NA and pET28a-M2 were successfully constructed and the corresponding fusion proteins were successfully expressed in BL21, respectively. Then, the expressed fusion proteins were purified and their immunogenicity was verified by Western blotting. The sequence analysis and expressions of these two genes laid a good foundation for further research on pathogenesis and diagnostic techniques of the avian influenza H9N2 subtype.
The purpose of this study was to construct recombinant plasmids of NA and M2 proteins and identify their expression. The NA and M2 genes of the H9N2 strains A/chicken/Shandong/LY1/2017 were amplified by PCR, respectively. Homology analysis was performed and the result confirmed that the two gene sequences had high homology with that of the current epidemic H9N2 strains. The NA gene was cloned into the prokaryotic expression vector pcold1 and the M2 gene was cloned into the prokaryotic expression vector pET28a. Recombinant plasmids of pcold1-NA and pET28a-M2 were successfully constructed and the corresponding fusion proteins were successfully expressed in BL21, respectively. Then, the expressed fusion proteins were purified and their immunogenicity was verified by Western blotting. The sequence analysis and expressions of these two genes laid a good foundation for further research on pathogenesis and diagnostic techniques of the avian influenza H9N2 subtype.
引文
[1] 田伟,仇铮,姜丽萍,等.禽流感病原学研究进展[J].中国动物保健,2014(2):31-34.
[2] H9N2亚型禽流感病毒的分离鉴定及其遗传进化分析[D].福州:福建农林大学,2015.
[3] 关立峥,王晶,卢昆鹏,等.两株H3N1亚型禽流感病毒(AIV)的生物学特性[J].农业生物技术学报,2017,25(7):1162-1169.
[4] Hayashi K,Kojima C.pCold-GST vector:a novel cold-shock vector containing GST tag for soluble protein production[J].Protein Expr Purif,2008,62(1):120-127.
[5] 周迎春,孙明,赵铁柱,等.禽流感病毒核蛋白基因的原核表达及其生物学活性分析[J].中国兽医杂志,2007,43(8):10-12.
[6] 李永仙,郑飞云,李崎,等.重组大肠杆菌BL21(DE3)-pET28a(+)-bgl诱导表达β-葡聚糖酶的条件优化[J].食品与生物技术学报,2009,28(2):250-255.
[7] 张珊珊,王志宇,卫吉芸,等.AIV的H6N2亚型NA与M2融合基因的原核共表达[J].畜牧与兽医,2012(Sup1):310.
[8] 马树杰,孔晖晖,施建忠,等.NA蛋白对流感病毒受体结合特性影响的研究[J].中国预防兽医学报,2016,38(7):518-522.
[9] Grantham M L,Stewart S M,Lalime E N,et al.Tyrosines in the influenza a virus M2 protein cytoplasmic tail are critical for production of infectious virus particles[J].J Virol,2010,84(17):8765.
[10] 张弟.重组FHL2的原核表达、纯化及抗血清的制备[D].南方医科大学,2008.
[11] Wang Y,Zhou L,Shi H,et al.Monoclonal antibody recognizing SLLTEVET epitope of M2 protein potently inhibited the replication of influenza A viruses in MDCKcells[J]].Biochem Biophys Res Commun,2009,385(1):118-122.
[12] van der Sande M A,Jacobi A,Meijer A,et al.The 2009 influenza A (H1N1) pandemic.Management and vaccination strategies in the Netherlands[J].Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz,2013,56(1):67-75.
[13] Shen Y,Wang X,Guo L,et al.Influenza A virus induces p53 accumulation in a biphasic pattern[J].Biochem Biophys Res Commun,2009,382(2):331-335.
[14] 刘江,何军,曾凡荣,等.2017年淮南市外环境H7N9病毒HA和NA基因特征分析[J].中国病毒病杂志,2018,8(1):50-57.
[15] 姜小华,仲人前,等.M2自身抗原及其三联体的克隆表达和初步鉴定[J].中华消化杂志,2001,21(9):530-533.
[16] 孙卫国.原核高效可溶性表达载体的构建及初步应用[D].北京:中国人民解放军军事医学科学院,解放军军事医学科学院,2005.
[17] 张强.SARS冠状病毒核蛋白的原核表达及初步应用的研究[D].太原:山西医科大学,2004.
[18] 郭元吉,温乐英,王敏,等.我国猪群中H9N2亚型毒株HA和NA基因特性的研究[J].中华实验和临床病毒学杂志,2004,18(1):7-11.
[19] 王泽霖,王川庆,赵军,等.H9N2亚型禽流感变异株致病性研究[C]//中国畜牧兽医学会禽病学分会第十六次学术研讨会,中国北京,2012.
[20] 田勇,郑雪花,张瑞华.禽流感病毒H9N2河北分离株HA和NA基因的遗传进化分析[J].黑龙江畜牧兽医,2012(23):74-77.
[21] 何伟.重组大肠杆菌表达包涵体药物蛋白的复性与纯化研究[D].上海:华东理工大学,2017.
[22] Zhu P,Liang L,Shao X,et al.Host cellular protein TRAPPC6AΔ interacts with influenza a virus M2 protein and regulates viral propagation by modulating M2 trafficking[J].J Virol,2016,91(1).pii:e01757-16.
[2] H9N2亚型禽流感病毒的分离鉴定及其遗传进化分析[D].福州:福建农林大学,2015.
[3] 关立峥,王晶,卢昆鹏,等.两株H3N1亚型禽流感病毒(AIV)的生物学特性[J].农业生物技术学报,2017,25(7):1162-1169.
[4] Hayashi K,Kojima C.pCold-GST vector:a novel cold-shock vector containing GST tag for soluble protein production[J].Protein Expr Purif,2008,62(1):120-127.
[5] 周迎春,孙明,赵铁柱,等.禽流感病毒核蛋白基因的原核表达及其生物学活性分析[J].中国兽医杂志,2007,43(8):10-12.
[6] 李永仙,郑飞云,李崎,等.重组大肠杆菌BL21(DE3)-pET28a(+)-bgl诱导表达β-葡聚糖酶的条件优化[J].食品与生物技术学报,2009,28(2):250-255.
[7] 张珊珊,王志宇,卫吉芸,等.AIV的H6N2亚型NA与M2融合基因的原核共表达[J].畜牧与兽医,2012(Sup1):310.
[8] 马树杰,孔晖晖,施建忠,等.NA蛋白对流感病毒受体结合特性影响的研究[J].中国预防兽医学报,2016,38(7):518-522.
[9] Grantham M L,Stewart S M,Lalime E N,et al.Tyrosines in the influenza a virus M2 protein cytoplasmic tail are critical for production of infectious virus particles[J].J Virol,2010,84(17):8765.
[10] 张弟.重组FHL2的原核表达、纯化及抗血清的制备[D].南方医科大学,2008.
[11] Wang Y,Zhou L,Shi H,et al.Monoclonal antibody recognizing SLLTEVET epitope of M2 protein potently inhibited the replication of influenza A viruses in MDCKcells[J]].Biochem Biophys Res Commun,2009,385(1):118-122.
[12] van der Sande M A,Jacobi A,Meijer A,et al.The 2009 influenza A (H1N1) pandemic.Management and vaccination strategies in the Netherlands[J].Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz,2013,56(1):67-75.
[13] Shen Y,Wang X,Guo L,et al.Influenza A virus induces p53 accumulation in a biphasic pattern[J].Biochem Biophys Res Commun,2009,382(2):331-335.
[14] 刘江,何军,曾凡荣,等.2017年淮南市外环境H7N9病毒HA和NA基因特征分析[J].中国病毒病杂志,2018,8(1):50-57.
[15] 姜小华,仲人前,等.M2自身抗原及其三联体的克隆表达和初步鉴定[J].中华消化杂志,2001,21(9):530-533.
[16] 孙卫国.原核高效可溶性表达载体的构建及初步应用[D].北京:中国人民解放军军事医学科学院,解放军军事医学科学院,2005.
[17] 张强.SARS冠状病毒核蛋白的原核表达及初步应用的研究[D].太原:山西医科大学,2004.
[18] 郭元吉,温乐英,王敏,等.我国猪群中H9N2亚型毒株HA和NA基因特性的研究[J].中华实验和临床病毒学杂志,2004,18(1):7-11.
[19] 王泽霖,王川庆,赵军,等.H9N2亚型禽流感变异株致病性研究[C]//中国畜牧兽医学会禽病学分会第十六次学术研讨会,中国北京,2012.
[20] 田勇,郑雪花,张瑞华.禽流感病毒H9N2河北分离株HA和NA基因的遗传进化分析[J].黑龙江畜牧兽医,2012(23):74-77.
[21] 何伟.重组大肠杆菌表达包涵体药物蛋白的复性与纯化研究[D].上海:华东理工大学,2017.
[22] Zhu P,Liang L,Shao X,et al.Host cellular protein TRAPPC6AΔ interacts with influenza a virus M2 protein and regulates viral propagation by modulating M2 trafficking[J].J Virol,2016,91(1).pii:e01757-16.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |