Genome Sequence Analysis Of Equine Influenza Virus H3N8 Subtype Inner Mongolia Strain And Prokaryotic Expression Of NS1 Gene

Equine influenza (EI) is an acute contagious respiratory infectious disease which is common for horses.In recent years, with the international horse racing increasing and the scope enlarging,it seriously threats horse racing because of the outbreak and epidemic ofequine influenza.It is very significant not only on virology and veterinary medicine study but also on prevent and control equine infuenza that master the epidemic regularity of EIV subtype H3N8 on molecular level.In this study. Virus of the local isolate A/equine/Inner Mongolia/8/08(H3N8) was propagated in SPF chicken embryo and nudeic acid was extracted. According to the conservation sequence of 3'terminus of eight gene fragment from EIV, a piece of primer was designed to reverse transcript RNA. Accordmg to the cDNA sequence from H3N8 EIV published in GenBank, fourteen pairs of specific primers were designed to amplify all of the eight gene by software Oligo6.0 and the products of RT-PCR was cloned into pMD18-T vector. The sequencing results show that all the cDNA has a complete open reading frame.By comparing with some strains of the same subtype published in GenBank,there is the highest percent homology of nucleotide with American strain Kentucky/02.The homology of nucleotide sequence of H A , N A , N P , N S 1 , N S 2 , M 1 , M 2 , P A , P B l a n d P B 2 i s 98.7%,99.0%,99.1%,99.5%,99.4%,97.5%,97.4%,98.7%,98.8% and 99.2% respectively,comparing with the sequence of the strain Kentucky/02.It showed that there is the same evolutional process of all the gene and this strain and the strain Kentucky/02 may derive from the same ancestor.The NS1 gene,700bp, was subcloned into prokaryotic expression vector pET-32a, and the positive recombinant plasmid was transformed into competence cell BL21 (DE3). The colony containing the positive plasmid pET-32a was screened and induced by 0.8mmol/L IPTG and analyzed by SDS-PAGE. The result in gel showed that the targat protein was mostly in inclusions when induced by IPTG,and the product was 46kDa fused with tag protein.The Western-blot result showed that the recombinant protein can specific bind with EIV(H3N8) positive serum.