Recombinant expression of human influenza A virus nucleocapsid protein and its antigen city analyses
10.3760/cma.j.issn.1003-9279.2008.03.017
- VernacularTitle:人流感病毒核蛋白的重组表达及其抗原性分析
- Author:
Yi-Hua BAO
1
;
Ruo-Lei XIN
;
Jie DENG
;
Fang WANG
;
Yuan QIAN
;
Jian-Xin WU
;
Ting ZHANG
Author Information
1. 首都儿科研究所
- Keywords:
Orthonyxoviridae;
Gene expression regulation,viral;
Antigens;
Immunohistochemistry;
Computational bidogy
- From:
Chinese Journal of Experimental and Clinical Virology
2008;22(3):208-210
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare anti-recombinant protein antibody from immunized mice with recombinant nucleocapsid protein (NP) of human influenza A3 (IFV-A3) virus expressed in prokaryotic cell, and to explore the feasibility of utilizing anti-recombinant protein antibody to detect influenza A virus. Methods NP genes of human influenza A virus were analyzed with computer softwares of ClustalX, Antheprot, et al. to determine the antigen city in conserved regions. Three different partial NP genes were harvested and cloned intopET-28(c) plasmid, the recombinant plasmids were induced to express partial NP segments in BL21 cells. The recombinant proteins were purified with Ni-agarose by affinity chromatography and immunized BALB/c mice. The polyclonal antisera harvested from mice were analyzed with Western Blot and immunohistochemistry assays to detect the reactions with IFV-A. Results Three recombinant plasmids were expressed with high yield in BL21 cells, about 15-20 mg/L. Western Blot Results indicated that the three prepared antis era (1:2000) positively reacted within from IFV-A3-infected cells. And immunohistochemistry assays suggested that anti-NP1, anti-NP2, anti-NP3 antisera positively reacted with IFV-A3 or IFV-Al-infected MDCK cells, with titers of 1:640 to 1:1280. Conclusion the recombinant NP of IFV-A3 would induce polyclonal antibodies with high titers in mice. The polyclonal antibodies would cress-react with IFV-A3 and IFV-AI. It is feasible to predict the antigen city with systematical bioinformatics analyses and then induce anti-IFV antibodies with high dilutions, and it is possible to be utilized in the early detection and sub typing analyses of IFV-infections.
