[禽流感病毒H5N1核蛋白的表达、纯化及宿主细胞体外相互作用蛋白的筛选]

[Expression and purification of avian influenza virus H5N1 NP protein, and screening interaction proteins of host cells in vitro].

作者信息

Wang Nai-Fu, Ma Jing, Zhang Xiao-Guang, Zhang Xiao-Mei, Bai Tian, Wang Min, Wen Le-Ying, Wang Da-Yan, Shu Yue-Long, Zhou Ling, Zeng Yi

机构信息

State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.

出版信息

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2010 Feb;24(1):27-9.

PMID:20848843

Abstract

OBJECTIVE

To express and purify H5N1 influenza virus (A/Anhui/1/2005) NP in prokaryotic system and to explore the NP-interacting proteins of human bronchial epithelial cells BEAS-2B in vitro.

METHODS

The full length H5N1 NP gene fragment was amplified by PCR, inserted into prokaryotic expression vector (pET30a) to generate NP expression plasmid pET30a-NP. After transforming pET30a-NP into E. coli (BL21), the expression of soluble NP protein was induced by IPTG. The expressed NP protein was purified by two steps with metal chelation chromatography and ion exchange chromatography. Then the total proteins of BEAS-2B cells was extracted for screening the components which have protein-protein interaction with purified NP by pull-down and LC-MS/MS methods.

RESULTS

The expression of H5N1 NP protein could be induced by IPTG in bacterial system using expression plasmid pET30a-NP. The soluble NP was purified. Twenty proteins were found by pull-down and LC-MS/MS, the further experiments may be needed to prove protein-protein interaction between them.

CONCLUSION

The soluble H5N1 NP fusion protein with high purity was obtained and twenty proteins were found which could interact with it by pull-down and LC-MS/MS.

摘要

目的

在原核系统中表达并纯化H5N1流感病毒（A/安徽/1/2005）核蛋白（NP），并在体外探索人支气管上皮细胞BEAS-2B中与NP相互作用的蛋白。

方法

通过PCR扩增H5N1 NP基因全长片段，将其插入原核表达载体（pET30a）构建NP表达质粒pET30a-NP。将pET30a-NP转化至大肠杆菌（BL21）后，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导可溶性NP蛋白表达。通过金属螯合层析和离子交换层析两步法纯化表达的NP蛋白。然后提取BEAS-2B细胞的总蛋白，采用下拉法和液相色谱-串联质谱（LC-MS/MS）法筛选与纯化的NP存在蛋白-蛋白相互作用的成分。