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


CONCLUSIONS
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.
Wang
Nai-Fu
NF
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.
Ma
Jing
J
Zhang
Xiao-Guang
XG
Zhang
Xiao-Mei
XM
Bai
Tian
T
Wang
Min
M
Wen
Le-Ying
LY
Wang
Da-Yan
DY
Shu
Yue-Long
YL
Zhou
Ling
L
Zeng
Yi
Y
D002460:Cell Line; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000378:metabolism*; D007251:Influenza, Human / Q000378:metabolism* / Q000821:virology; D019590:Nucleocapsid Proteins; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014758:Viral Core Proteins