Cloning and Expression of Neuraminidase Gene of H5N1Subtype Avian Influenza Virus

SONG Jian-ling; ZHANG Fu-qiang*; WANG Jun-ping; HU Yuan-yuan

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February 2006

SONG Jian-ling, ZHANG Fu-qiang*, WANG Jun-ping and HU Yuan-yuan. Cloning and Expression of Neuraminidase Gene of H5N1Subtype Avian Influenza Virus[J]. Virologica Sinica, 2006, 21(1): 43-46.

Citation: SONG Jian-ling, ZHANG Fu-qiang*, WANG Jun-ping, HU Yuan-yuan. Cloning and Expression of Neuraminidase Gene of H5N1Subtype Avian Influenza Virus .VIROLOGICA SINICA, 2006, 21(1) : 43-46.

H5N1亚型禽流感病毒神经氨酸酶基因的克隆与表达

1.
云南省热带亚热带动物病毒病重点实验室，云南昆明 650224

摘要

根据已知H5N1亚型禽流感病毒神经氨酸酶基因（na）序列设计、合成克隆引物。自H5N1亚型病毒感染的鸡胚尿囊液中提取总RNA，反转录后采用高可信度DNA聚合酶（PyobestTM DNA Polymerase）扩增na基因，采用Invitrogen定向表达系统（ChampionTM pET directional TOPO expression system）进行克隆表达，纯化获得N末端携带多聚组氨酸标签的重组神经氨酸酶，分子量约53.8kDa。分析重组NA的免疫反应性和免疫原性，结果表明：重组NA能与H5N1亚型病毒抗血清发生特异性结合，且其免异动物后能诱导机体产生特异性抗体，具有良好的抗原性。关键词：禽流感病毒；H5N1亚型；神经氨酸酶；表达
- 禽流感病毒
- , H5N1亚型
- , 神经氨酸酶
- , 表达

Cloning and Expression of Neuraminidase Gene of H5N1Subtype Avian Influenza Virus

1.
Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Kunming 650224, China

Abstract

A pair of clone primers were designed and synthesized based on neuraminidase(NA) gene sequences of known Avian influenza virus H5N1 subtypes. NA gene were amplified from total RNA, which had been extracted from allantoic fluid of H5N1 subtype virus inoculated embryo, by reverse transcriptase-polymerase chain reaction using high proofreading polymerase (PyobestTM DNA Polymerase), and expressed using Invitrogen championTM pET directional TOPO expression system. 53.8kDa recombinant NA fusion with polyhistidine (6xHis) tag in N-terminal was expressed and purified. Its immunoreactivity and immunogenicity had been analyzed. The results showed the recombinant NA could not only bind to antiserum against H5N1 subtype virus with specificity but also elicit specific antibody in immunizing animal, and possessed good antigenicity.
- Avian influenza virus
- , H5N1 subtype
- , Neuraminidase
- , Expression

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Cloning and Expression of Neuraminidase Gene of H5N1Subtype Avian Influenza Virus

1. Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory, Kunming 650224, China

Abstract: A pair of clone primers were designed and synthesized based on neuraminidase(NA) gene sequences of known Avian influenza virus H5N1 subtypes. NA gene were amplified from total RNA, which had been extracted from allantoic fluid of H5N1 subtype virus inoculated embryo, by reverse transcriptase-polymerase chain reaction using high proofreading polymerase (PyobestTM DNA Polymerase), and expressed using Invitrogen championTM pET directional TOPO expression system. 53.8kDa recombinant NA fusion with polyhistidine (6xHis) tag in N-terminal was expressed and purified. Its immunoreactivity and immunogenicity had been analyzed. The results showed the recombinant NA could not only bind to antiserum against H5N1 subtype virus with specificity but also elicit specific antibody in immunizing animal, and possessed good antigenicity.