Expression of M1 protein of Avian Influenza Virus and Analysis of Its Immunoreactivity

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

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October 2005

SONG Jian-ling, WANG Jin-ping, HU Yuan-yuan and ZHANG Fu-qiang*. Expression of M1 protein of Avian Influenza Virus and Analysis of Its Immunoreactivity[J]. Virologica Sinica, 2005, 20(5): 515-518.

Citation: SONG Jian-ling, WANG Jin-ping, HU Yuan-yuan, ZHANG Fu-qiang*. Expression of M1 protein of Avian Influenza Virus and Analysis of Its Immunoreactivity .VIROLOGICA SINICA, 2005, 20(5) : 515-518.

禽流感病毒M1蛋白的表达及其免疫反应性分析

1.
云南省热带亚热带动物病毒病重点实验室云南昆明

摘要

根据已发表的禽流感病毒M1基因序列设计合成PCR克隆引物,自接种H5N1亚型病毒的鸡胚组织中提取RNA,反转录后采用高可信度DNA聚合酶(PyobestTMDNA Polymerase)经PCR扩增M1基因,采用Invitrogen定向表达系统(ChampionTMpET 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蛋白,分子量约29.8 kDa。采用单克隆抗体和阳性血清经ELISA、阻断ELISA、免疫印迹分析重组蛋白的免疫反应性。结果发现:重组M1蛋白能与单克隆抗体和阳性血清发生特异性结合,且此结合能被天然病毒抗原阻断。研究表明:重组蛋白M1具有良好免疫反应性。
- 禽流感病毒
- , M1蛋白
- , 表达
- , 免疫反应性

Expression of M1 protein of Avian Influenza Virus and Analysis of Its Immunoreactivity

Abstract

A pair of primers were designed based on M1 gene sequence of known H5N1 Avian influenza virus.M1 gene was cloned from total RNA,extracted from tissue of H5N1 subtype virus inoculated embryo by reverse transcriptase-polymerase chain reaction using high proofreading polymerase(Pyobest~TM DNA Polymerase),and expressed using Invitrogen champion~TM pET directional TOPO expression system.Recombinant protein containing polyhistidine(6xHis) tag in N-terminal about 29.8kDa in size,wac obtained and purified.Its immunoreactivity was analyzed by Western blot,ELISA and blocking ELISA using MAb and positive serum.The results showed recombinant M1 protein can bind to MAb and positive serum with specificity,and this binding reactivity can be blocked by natural viral antigen.This indicates that the recombinant M1 protein possesses good immunoreactivity.
- Avian influenza virus
- , M1 protein
- , Expression
- , Immunoreactivity

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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

Expression of M1 protein of Avian Influenza Virus and Analysis of Its Immunoreactivity

Abstract: A pair of primers were designed based on M1 gene sequence of known H5N1 Avian influenza virus.M1 gene was cloned from total RNA,extracted from tissue of H5N1 subtype virus inoculated embryo by reverse transcriptase-polymerase chain reaction using high proofreading polymerase(Pyobest~TM DNA Polymerase),and expressed using Invitrogen champion~TM pET directional TOPO expression system.Recombinant protein containing polyhistidine(6xHis) tag in N-terminal about 29.8kDa in size,wac obtained and purified.Its immunoreactivity was analyzed by Western blot,ELISA and blocking ELISA using MAb and positive serum.The results showed recombinant M1 protein can bind to MAb and positive serum with specificity,and this binding reactivity can be blocked by natural viral antigen.This indicates that the recombinant M1 protein possesses good immunoreactivity.