[Expression and identification of H5 subtype hemagglutinin of avian influenza A virus in insect cells].
OBJECTIVE
To 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.


METHODS
H5 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.


RESULTS
The 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.


CONCLUSIONS
The 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.
Wen
Kun
K
Central Laboratory of Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Qiu
Li-Wen
LW
Wang
Ya-di
YD
Che
Xiao-Yan
XY
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004912:Erythrocytes / Q000166:cytology / Q000276:immunology; D005822:Genetic Vectors / Q000235:genetics*; D006168:Guinea Pigs; D006386:Hemagglutination Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology; D018411:Spodoptera; D014162:Transfection
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins; C109963:hemagglutinin, avian influenza A virus