Expression and identification of H5 subtype hemagglutinin of avian influenza A virus in insect cells.
- Author:
Kun WEN
1
;
Li-Wen QIU
;
Ya-di WANG
;
Xiao-Yan CHE
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Baculoviridae; genetics; Cell Line; Erythrocytes; cytology; immunology; Genetic Vectors; genetics; Guinea Pigs; Hemagglutination Tests; Hemagglutinin Glycoproteins, Influenza Virus; biosynthesis; genetics; immunology; Influenza A Virus, H5N1 Subtype; genetics; immunology; Recombinant Proteins; biosynthesis; immunology; Spodoptera; Transfection
- From: Journal of Southern Medical University 2007;27(1):20-23
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo clone and express avian influenza A virus [A/Hong Kong/482/97(H5N1)] H5 subtype hemagglutinin in baculovirus-insect cell expression system and investigate the antigenicity and bioactivity of the recombinant protein.
METHODSH5 gene of influenza A virus was amplified by PCR. The recombinant bacmid was obtained by cloning the gene to the donor plasmid of pFastBacHTB and transformed into DH10Bac competent cells. The recombinant baculovirus stock was prepared by transfecting the recombinant bacmid DNA into the insect cell line for protein expression after amplification. Immunofluorescene assay (IFA) and Western blotting were performed to identify the antigenicity of the recombinant protein, and hemagglutination assay was used to identify its bioactivity.
RESULTSThe recombinant his-H5 protein was expressed in the insect cells with a relative molecular mass of 64,000, which showed erythrocyte-agglutinating activities with the red blood cells of guinea pig. Western blotting and IFA demonstrated that the recombinant his-H5 could be recognized and bound by standard anti-H5 sera.
CONCLUSIONThe recombinant his-H5 with a post-translation modification is successfully obtained in insect cells, which may provide a potential source for further study of the antigen's biological function and for production of the subunit vaccine or monoclonal antibodies.
