Expression Of HA, NA And M2 Genes Of Avian Influenza Virus A/Goose/Guangdong/1/96(H5N1)

Avian Influenza(AI),is an infection and/or disease syndrome caused by type A influenza virus. It is widely spread in many countries and areas throughout the world, and caused great economic losses in poultry industry. The main characteristics of the highly pathogenic avian influenza (HPAI) are high mortality, frequently causes the chicken group completely die, and it has been classified as A level virulent infectious disease by O.I.E. The 97'events happened in Hong Kong that highly pathogenic avian influenza (HPAI) H5N1 subtype infected human directly, especially caused them dead, and 99'events that H9N2 virus isolated from two Hong Kong children with slight flu symptom made the infection draw much attention in the public Hygiene field. In the Year 2001 ,Hong Kong isolated the H5N1subtype of HPAIV again. Special zone government invested 800 million Hong Kong dollars to slaughter the12 million chickens in Hong Kong,but, up to now there is no effective method to thoroughly purify the living poultry market which polluted by H5N1 of HPAIV. From above, we can know it is necessary to study this infection to protect our human's benefits. Hence expressing HPAIV's relevant protein is important to prepare sub-unit vaccine for providing the poultry suffers from the infection, And prepare molecular diagnosis kits, study the relationship between crystalline structure of protein and pathogenic mechanism. Immunogenicity relevant protein of Avian flu virus include HA ,NA,NP and M2 four kinds of proteins. The HA is one kind of protein that is located in the surface of virus envelope which can deduce the poultry organ produce neutralization antibody; the NA is another target antigen of AIV's humoral immunity, it may causes organ produce the specific antibody, yet it does not has the virus neutralization function; the NP can only produce very feeble capability against infection; the M2 takes the relatively significant role in the cross immunity.At the present,AIV Immunogenicity relevant protein expressing systems mainly are the colon bacillus expressing system ,the insect expressing system,yeast expressing system, but each method has defect.Hence ,this paper based on the relevant references ,analyzed the NA ,HA ,and M2 gene and protein of AIV H5N1 subtype utilized the pichia pastoris, baculovirus / insect ,and GST fuse expressing system to express the AIV Immunogenicity relevant protein1 To construct the recombinant pPIC9KNA vector, the NA gene of AIV was amplified by RT-PCR, and then cloned into the plasmid vector pMD-18T after that the complete coding region of NA gene of AIV was subcloned into the pichia expression vector pPIC9K with the AOX1 promoter and α-factor signal sequence between NotⅠ and SnaBⅠ site . The recombinant plasmid pPIC9KNA was transformed into pichia pastoris strain GS115 by the method of Electroporation. Muti-copy recombinants were screened on concentrations of G418 and Mut＋ phenotype was confirmed .Mutiple insert transformants were fermented in flasks and inducted with 1% methanol. After four days of methanolinduction, the supernatant was estimated to detect the biological activity of NA gene. AGP, NA and Western-blot assays proved it having good antigenicity and high specificity .It set a good base for research and development of the AIV subunit vaccine. 2 The complementary DNA of 1.7 kb HA gene was prepared from the viral gene RNA by RT-PCR. The amplified fragment was cloned into the plasmid vector pMD18-T and then sequenced. Compare the HA sequence with 3 published H5N1 subtype's, the nucleotide homology are 99﹪,97.9﹪and 98.1﹪ respectively .The signal sequence of coding HA protein was deleted, then subcloned into the downstream of the baculovirus transfer vector pMelBacA that contain the honeybee melittin secretion signal and named as pMelH5HA. The pMelH5HA was sequenced and co-transfected the sf9 cell with the Bac-N-BlueTM DNA by the t...