Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2010, Vol. 30 Issue (01): 12-18    DOI:
    
Expression of the Hemagglutinin and Neuramidinase Gene of Influenza A Virus H5N1 in E.coli
ZOU Shu-mei1,YU Zai-jiang1,ZHANG Ye1,XIN Li1,CHEN Yong-kun1,TANG Qi-hui2,CHEN Yu-bao3,CHEN Qing-xuan2,SHU Yue-long1
1. Institute for Viral Disease Control and Prevention,China National Influenza Center,Beijing 100052,China
2.Beijing Biokit Inc.,Beijing 100094,China
3.Beijing Sinogreen Institute for Bioeconomy,Beijing 102206,China
Download: HTML   PDF(707KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Objective:To clone, express and characterize the HA（hemagglutinin） and NA（neuramidinase，NA） protein  of avian influenza virus H5N1. Methods:On the basis of successful clone the full length HA and NA gene and sequence analysis of avian influenza virus H5N1,Ligated part of the gene into pET32a（＋）.full of the gene into pGEX4T-1.An expression vector pET32a（＋）/HA(49~1587bp),pET32a（＋）/NA(121~1141bp),pGEX4T-1/HA、pGEX4T-1/NA were constructed and expressed in E.coli BL21/rosetta induced by IPTG. recombinant protein was purified through Ni2＋ and GSTrap 4B affinity chromatography column. Western blotting and ELISA were used to determine the antigenic of the recombinant protein. Results:The recombinant capsid gene can be overexpressed in E.coli. SDSPAGE showed that the gene could express product as same as I might expect. The purity of the protein is greater than 90%.ELISA and Western blotting showed that the recombinant protein has good antigenic. Conclusion:The HA and NA protein  of avian influenza virus H5N1 has been successful cloned and expressed, which could be useful for developing diagnose reagents or vaccine of H5N1.

Key words： avian influenza virus H5N1      hemagglutinin      neuramidinase      clone and express     
Received: 12 November 2009      Published: 27 January 2010
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
JU Chu-Mei
XU Zai-Jiang
ZHANG Ye
XIN Li
CHEN Yong-Kun
TANG Qi-Hui
CHEN Yu-Bao
CHEN Qing-Han
SHU Ti-Long
Cite this article:

JU Chu-Mei, XU Zai-Jiang, ZHANG Ye, XIN Li, CHEN Yong-Kun, TANG Qi-Hui, CHEN Yu-Bao, CHEN Qing-Han, SHU Ti-Long. Expression of the Hemagglutinin and Neuramidinase Gene of Influenza A Virus H5N1 in E.coli. China Biotechnology, 2010, 30(01): 12-18.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2010/V30/I01/12

［1］ Christensen J P,Dietz,H H,Bisgaard M. Phenotypic and genotypic characters of isolates of Pasteurel la multoci da obtained from backyard poultry and from two outbreaks of avian cholera in avifauna in Denmark. Avian Pathol,1998,27 :373381. 
［2］ Berman S M, Hirsh D C. Partial Characterization of Plasmids from Pasteurel la multoci da isolated from turkeys. Antimicroblal Agents Chemotherapy,1978,14 :348352. 
［3］ Subbarao K, Klimov A, Katz J, et al . Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness .Science,1998,293 :393396. 
［4］ Zhou N N, Short ridge K F, Claas E C J,et al . Rapid evolution of H5N1 influenza viruses in chicken in Hongkong. J Virol, 1999,73 :33663374. ［5］ Alexander D J . A review of avian influenza in different bird speices . Veterinary Micorbiology,2000,74:313. 
［6］倪建强,张春玲,李海燕,等. 猪流感病毒核蛋白基因的原核表达及其在诊断中的初步应用. 中国预防兽医学报, 2004, 26(1) : 18  21. Ni J Q,Zhang C L,Li H Y,et al.Chinese Journal of Preventive Medicine,2004,26(1):1821. 
［7］丛彦龙,刘金华. 流感病毒血凝素在宿主特异性转变种的分子基础. 中国兽医学报, 2008, 28 (6) : 737742. Cong Y L,Liu J H.Chinese Journal of Veterinary Science,2008,28(6):737742. 
［8］ Webster R G,BeanW J,Gorman O T,et al . Evolution and ecology of influenza A viruses . Microbiol Rev,1992,56 :152179. 
［9］ Alexander D J . A review of avian influenza in different bird speices.Veterinary Micorbiology,2000,74:313. 
［10］ Pereira H G, Tumova B,Law V G. Animal influenza A virus. World Health Organization,1965,32:855860 
［11］ 费东亮,金宁一,马鸣潇,等. H5亚型禽流感病毒HA基因在重组杆状病毒中的表达及检测. 中国兽医学报,2006,26 (4) :387389. Fei D L,Jin N Y,Ma M X,et al.Chinese Journal of Veterinary Science,2006,26(4):387389. *
［12］ 施源,袁渭康,陈敏恒. 不稳定的基因工程酵母生长动力学研究. 生物工程学报, 1990, 6 (1) : 4449. Shi Y,Yuan W K,Chen M H.Chinese Journal of Biotechnology,1990,6(1):4449. 
［13］ Claas E C,Osterhaus A D,Von Beek R, et al. Human influenza H5N1 virus related to a highly pathogenic avian influenza virus. Lancet, 1998, 35 (1) : 472477. 
［14］ Colman P M,Varghese J N,Laver W G. St ructure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature,1983,303 (5912) :4144. 
［15］ 宋建领,张富强,王金萍,等. H5N1 亚型禽流感病毒神经氨酸酶基因的克隆与表达.中国病毒学,2006,21(1):4346. Song J L,Zhang F Q,Wang J P,et al.Virologica Sinica,2006,21(1):4346. 
［16］ 郑其升,刘华雷,张晓勇,等. H9N2 亚型AIV HA 基因的原核表达及间接ELISA 方法的建立.中国病毒学, 2005, 20 (3) :293297. Zheng Q S,Liu H L,Zhang X Y,et al.Virologica Sinica,2005,20(3):293297. 
［17］ 南文龙，金宁一，鲁会军,等.H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析.中国免疫学杂志，2009，7(25)：630637. Nan W L,Jin N Y,Lu H J,et al.Chinese Journal of Immunology,2009,7(25):630637. 
［18］ 钟晓琴.感染人类的禽流感病毒A ( H5N1) 研究进展.中华肺部疾病杂志，2009，2（1）6375. Zhong X Q.Chinese Journal of Lung Disease,2009,2(1):6375.
[1] YUAN Ying-shuo, SONG Fei-fei, LIU Guo-qiang, LIU Ye, ZHANG Shou-feng, ZHANG Le-cui, HU Rong-liang. Construction and Immune Efficacy of Recombinant Adenovirus Expressing H Gene of Canine Distemper Virus[J]. China Biotechnology, 2014, 34(1): 64-70.
[2] YAO Jing, REN Jing, WU Zheng-jun, SUN Ke-jie, GUO Ben-heng. Cloning and Expression of the Key Enzyme Gene in Biosynthesis of Sialyl Lewis X[J]. China Biotechnology, 2011, 31(12): 51-56.
[3] YE Ruo-song, LI Peng, ZHANG Zhao-lin, WAN Cui-xiang, CUI Jia, WEI Hua. Cloning, Expression and Function Analysis of Serpin Gene in Bifidobacterium bifidum[J]. China Biotechnology, 2011, 31(02): 38-42.
[4] HUANG Jin-song, HUANG Jie-qin, TANG Qi-hui, CHEN Yu-bao. Expression and Application Research of the C-terminal Gene Fragment of P1 Protein of Mycoplasma pneumoniae in E.coli[J]. China Biotechnology, 2010, 30(11): 65-69.
[5] LEI Xiu-Ping, CHEN Hua-Jun, LIU Yong, TANG Qi-Hui, CHEN Yu-Bao, LI Wen-Zheng. Expression of the Glycine-ich RNA-binding Protein of Tobacco in E.coli[J]. China Biotechnology, 2010, 30(08): 27-30.
[6] . Expression Of The Hemagglutinin And Neuramidinase Gene Of Influenza A Virus H9N2 in P.methanolica[J]. China Biotechnology, 2010, 30(07): 0-0.
[7] ZHANG Li-Na . The Comparisons of cloning，expression and immunogenicity of conservation region fragment of HN gene among different NDV genotypes[J]. China Biotechnology, 2008, 28(5): 93-98.
[8] . Characterization of HA Gene and NA Gene of A pigeon H5N1 Influenza A Virus[J]. China Biotechnology, 2006, 26(05): 17-21.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会