Expression of neuraminidase gene of influenza virus H1N1 in baculovirus-expression system
10.3760/cma.j.issn.1003-9279.2013.02.001
- VernacularTitle:H1N1亚型流感病毒NA基因在昆虫杆状病毒系统中的表达及鉴定
- Author:
Li-Hong YAO
1
;
Jin-Qi FU
;
Ai-Jun CHEN
;
Xiao-Yu LIU
;
Peng-Wei XU
;
Jian-Qiang GUO
;
Le-Cui ZHANG
Author Information
1. 中国疾病预防控制中心病毒病预防控制所
- Keywords:
Orthomyxoviridae;
Neuraminidase;
Baculovirus;
Cells,cultured
- From:
Chinese Journal of Experimental and Clinical Virology
2013;27(2):81-84
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant baculovirus with NA gene of Influenza H1N1 virus.Methods Full-length NA gene of Influenza virus H1N1 (A/PR/8/34) was amplified by PCR and inserted into pFastBacdual vector to construct the recombinant baculovirus transfer vector pFBD-NA.Recombinant shuttle vectors rBacmid-NA was then obtained after transforming DH10B competent cells containing bacmid plasmids.After transfecting into sf9 cells,recombinant baculovirus rBac-NA was obtained.The rBac-NA genome was extracted and identified by PCR.NA protein expressed by recombinant baculovirus-infected sf9 cells was determined by IFA,Western Bolt and ELISA.Results PCR results proved that recombinant shuttle vectors rBacmid-NA was successfully constructed.NA protein was detected by IFA and showed strong specific green fluorescence on the surface of infected cells.NA protein was recognized by two polyclonal antibodies specific for NA in Western Blot.ELISA showed specific reaction of recombinant NA protein with mouse polyclonal antibody against influenza virus (PR8),indicating high antigenicity.Conclusion Recombinant baculovirus rBac-NA that expresse NA protein of influenza virus was successfully constructed.This work provides a basis for further study on NA protein function and novel influenza vaccine development.
