The HA Gene of H5N1 Avian Influenza Virus Expressed in Insect Cells and Its Biological Activity
- VernacularTitle:H5N1亚型禽流感病毒HA基因在昆虫细胞中的表达及其生物活性鉴定
- Author:
Xiao-Ji ZHANG
;
Ming LIU
;
Chun-Guo LIU
;
Tao YANG
;
Yun ZHANG
;
- Publication Type:Journal Article
- Keywords:
Avian influenza virus H5N1 HA Baculovirus expression
- From:
China Biotechnology
2006;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
The 1.7 kb full-length hemagglutinin (HA) gene fragment of H5N1 subtype avian influenza virus was amplified by RT-PCR and then cloned into the pFastBacHT donor plasmids. The recombinant plasmid pFastBac-H5 was identified by restriction enzyme digestion and sequencing. Following the transposition pFastBac-H5 into the bacmid in DH10Bac E.coli competent cells, the colonies were identified by blue and white selection. The recombinant bacmid (rBacmid-H5) was verified by PCR analysis. Transfection of rBacmid-H5 DNA into sf9 cells using Cellfectin reagent results in the production of recombinant viral stock. Cells were harvested 72h post infection and analyzed by SDS-PAGE, Western-blot, hemagglutination and hemagglutination inhibition test;the expressed HA protein (rH5)shows hemaggluting activity and can be inhibited by H5N1 virus immunized chicken sera. On the other hand, immunization of chickens with rH5 protein results in high titers of H5N1 virus specific hemagglutation inhibition antibodies, which proved its biological activity.
