Generation Of A Reassortant A/HNP/03(H₉N₂) Influenza A Virus By 12 Plasmid-based Transfection System

In this study , the RNA segments of HA and NA gene were extracted from a donor strain ,A/Chicken/HN43/98(H9N2), which was isolated from Henan province and indentified in 1998 ;then were transcripted reversely into cDNA by RT-PCR technology ,and then the two pairs of primers of HA and NA gene were phosphated respectively . Consequently, HA and NA gene were amplified by the cDNA used as template. Then the segments of HA and NA gene amplified were digested respectively by T4 DNA polymerase . The plasmid vector pPolâ… Sap â… T were digested by the restriction enzyme Sap â…  and DNA polymerases and so on. At first , The plasmid pPolâ… Sap â… was digested by the restriction enzyme Sap â… ,then was complemented by Klenow DNA polymerase , and then the phosphate group of the 5' termination was removed by Calf Intestine Alkaline Phospatase (CIAP).Linear plasmid vector pPolâ… Sap â… T treated linked respectively up the cDNA of HA and NA gene descriped above with the catalysis of T4 DNA ligase in vitro ,then the positive plasmids pPolâ… HAT and pPolâ… NAT were selected respectively.The positive plasmids pPolâ… HAT and pPolâ… NAT reassorted successfully in the study and other vRNAs transcription plasmids of the transfection system as well as the protein plasmids (pcDNA3.0-PB1,pcDNA3.0-PA,pcDNA3.0-NP,pcDNA3.0-PB2) from the transfection system coincubated for an hour with the reagent for transfection ,then cotransfected into near-cofluent 293T cell.Nine-to-11-day-old specific pathogen-free (SPF) embryonated chicken embryos were inoculated with tissue culture fluids of posttransfection , then the allantoic fluids of embryonated-eggs were harvested , HA test was carried out ,and the result was 4log2. Then the reassortant AIV were passaged three times in SPF embryonated chicken embryos. HA test was performed and the result was up to 8log2. The success of reassortment of H9N2 subtype Influenza A Virus was confirmed by the identification of serology and molecular biology.In this experiment , the local isolate A/Chicken /HN43/98 (H9N2) AIV from Henan province was used to be a donor of HA and NA gene , then a HNP/03(H9N2) Influenza A Virus strain was reassorted successfully. This do not only make a basis for further research of the mechanism for virulence variation of H9N2 subtype Influenza A Virus and biological function, but also present a kind of model for the development of good vaccine candidate of Influenza A Virus.