HA And HA1 Of AIV H7 Subtype Expressed In Recombinant Baculovirus And Detection Of Expressed Products Antigenicty

Avian influenza(AI)is caused by type A avian influenza virus,a member of Orthomyxoviridae.Avian influenza with low virulence is characterized by respiratory symptom and lowproduction.The highly pathogenic AIV once outbreak,will cause fairly severity economic damageto China and even many worldwide poultry countires.Contrast to H5 AIV, the outbreak of H7 AIVwill also cause great damage to poultry.The H7 AIV not only make incursions into chicken, duckbut also pathopoiesia bird(H7N1),and even infect humans. The AIV genome which encodes 10proteins related to the viral structure and function is comprised of eight negative-strand RNA.,andthe hemagglutinin (HA) was the main protein of surface spikes. The hemagglutinin (HA) is avirulence-associated glycoprotein. The hemagglutinin can induce neutralizing antibodies in vivo.Therefore, the hemagglutinin plays an important role in diagnose and control avian influenza.In this study, I took pMD18-T-H7-HA conserved in our lab as template,removal nucleotidewhich encode signal tide. Using Primer5.0 and Oligo4.0,respectively design HA and HA1 twoprimers.The HA and HA1 gene were amplied by PCR from pMD18-T-H7-HA. The PCR productwas respectively inserted into Sacâ… and Kpnâ… MCS of the vector pBlueBachis2A.They weredesignated as pBlueBacHis-H7-HA and pBlueBacHis-H7-HA1.Purified pBlueBacHis-H7-HA andpBlueBacHis-H7-HA1 and Bacmid-N-blue DNA were cotransfected into insect cell Sf9,thenrecombinant baculovirus named P1 was harvested after 5d. P1 was on monolayer Sf9 after the thirdplaque purification to reproduce in quantity, which was named P2. After P2 was inoculated on Sf9monolayer cell 4d the CPE cells were harvested.The SDS-PAGE analysis showed that HA and HA1gene were expressed in recombinant baculovirus, molecular weight is about 66ku and 42ku.Westernblotting and Dot-ELUSA analysis proved that the recombinant protein had good reactiveability against H7 subtype serum and did not have cross reaction against chicken H5 and H9antiserum. The hemagglutinin and indirect immunofluorescence ceterified that HA and HA1 hadgood reactivity.The expressed H7 recombinant HA and HA1 protein which took use of bacilovirus systemwas soluble,6 His-tag was easily purified.The recombinant HA and HA1 had good reactivity. Theresults showed that the recombinant H7-HA and HA1 gene was specifically expressed in Sf9 cellswith the expected sizesand the expression proteins had immunoreactivity in immunoblot assay.