Gene Analysis And Serological Investigation Of Avian Influenza N6 Subtype In Guizhou Province

Avian influenza(AI)is a highly contagious infectious disease infected by many kinds of animals by Avian influenza Virus(AIV).NA is an important antigen on the surface of avian influenza virus particles,which plays an important role in the process of virus replication.In recent years,the outbreak of avian influenza H5N6 subtype and the infection in human have been reported,so avian influenza H5N6 subtype has begun to be concerned.At present,avian influenza viruses N6 subtype reported in China include H3N6,H4N6,H5N6 and H6N6.In our laboratory,the avian influenza virus H6N6 subtype was isolated from the clinical healthy ducks.In this study,the the NA gene of the avian influenza virus H6N6 subtype Guizhou strain was cloned and sequenced,and expressed in the prokaryotic cells and used for the preparation of the polyclonal antibody and the serological investigation of the N6 subtype avian influenza,which provides the basic data for prevention and control of the N6 subtype avian influenza in Guizhou Province.1.Cloning and sequence analysis of avian influenza virus N6 geneAccording to N6 gene sequence of avian influenza virus in Gen Bank(NO.KJ484613),the specific primers were sesigned and synthesized,and used for amplification and sequence anlysis of the N6 gene of avian influenza virus isolated from Guizhou prevince.The results showed that the coding region of the N6 gene were 1380 bp in 4 strains of H6N6 subtype avian influenza virus,which could encode 459 amino acids,from which lack 33 nucleotides from 175 to 207 sites,there are 11 amino acid deletion(TNSTTTIINN)in the neck of the encoded protein.The nucleotide homology of the N6 gene in Guizhou strain is 96.0%~99.1%,of which A/duck/Guizhou/01/2017(H6N6)and A/duck/Guizhou/02/2017(H6N6)are the same branch,but A/duck/ Guizhou/03/2017(H6N6)and A/duck/ Guizhou/04/2017 are the other branch.The N6 subtype strain were obviously located in different branche with the non-N6 subtype strains.2.Prokayotic expression of NA gene and preparation of polyclonal antibodyThe amplification products of N6 gene of A/duck/Guizhou/02/2017 strain were reclaimed and cloned into prokaryotic expression vector PET-32a(+),and conversed into E.coli BL21(DE3)and expression induced by IPTG.The expressed protein were purified by affinity chromatography of His-tag column,and used for preparation of polyclonal antibody in rabbit,then the antigenicity of the N6 gene product were detected by Western-blot.The results showed that the expression form of N6 gene was mainly inclusion body,which the molecular weight of the expressed protein was about 70 k Da.the recombinant protein were identified by His antibody and NA antibody from rabbits.3.Serological survey of N6 subtype Avian influenza in Guizhou ProvinceUsing the prokaryotic expression N6 protein as the envelope antigen,813 of the chicken serum samples,which collected in chicken farms from 2016 to 2017,were detected by ELISA.The results showed that 186 of the chicken serum samples were positive,which the positive rate was 22.88%(186/813),in which the positive rate was 9.76%(20/205)in 2016,and27.30%(166/608)in 2017.These results indicated the positive rate was increased in the N6 subtype avian influenza.In summary,the N6 gene of avain influenza virus were cloned and sequenced,and expressed in the prokaryotic cells and the polyclonal antibody of N6 protein were preparated,and the N6 subtype antibody were detected and analyzed,which suggested that the infection of N6 subtype avian influenza has a rising trend in Guizhou province.These results provides the scientific data for the prevention and control of N6 subtype avian influenza in Guizhou Province.