Cloning and expression of hemagglutinin gene of H5N1 subtype avian influenza virus

A pair of clone primers was designed and synthesized based on hemagglutinin(HA) gene sequences of known H5N1 subtype avian influenza viruses.HA gene were amplified from total RNA,which had been extracted from inactivated Yunnan H5N1 subtype virus positive clinical tissue sample,by reversing transcriptase-polymerase chain reaction using high proofreading polymerase(Pyobest TM DNA Polymerase),and was expressed using Invitrogen Champion TM pET directional TOPO expression system.Recombinant HA containing polyhistidine(6xHis) tag in N-terminal is about 78 ku in size,which had been obtained and purified.Its immunoreactivity was analyzed by western blot and ELISA using positive serum.The results showed recombinant HA can bind to antiserum against H5N1 subtype virus with specificity and possesses good immunoreactivity.