Cloning, Sequence Analysis And Construction Of Eukaryotic Expression Plasmids For NS1 Gene Of Swine Influenza Virus (H3N2)

Swine influenza virus( SIV) is the infectious pathogen, which causes disease with changes of pigs'respiratory organs. The genome of SIV are composed of 8 segmented negative-strand RNA and encode 10 proteins. The protein encoded by NS1 gene is the only nonstructural protein in the 10 proteins. The NS1 protein plays an important role in some fields because of its conservatism and antigenicity , such as differential diagnosis between natural infection and vaccine immune,epidemic survey and forecast of influenza .The isolate of A/Swine/Guangdong/99(H3N2)was made as studying strain. The NS1 through encoding cDNA of the isolate was amplified and cloned. The virus RNA was directly extracted and purified from chicken embryo allantoic fluid. According to published gene sequence,a pair of specific primers to the NS1 gene was designed and synthesized. After that ,the NS1 cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Then the complete NS1 gene was sequenced. The results showed that the cloned 778 bp fragment covered the whole NS1 gene including the completely open reading frame(ORF).When comparing with the other eleven strains that were downloaded from www.flu.lanl.gov,the homology of nucleotide sequence ranged from 91.7% to 96.8 % and homology of deduced amino acid was from 90.4% to 96.8%.These indicated that NS1 genes of swine influenza viruses were well conserved. Comparison of the amino acid sequences of NS1 genes of the isolate with the other eleven strains demonstrated that nonstructural(NS1)protein of the isolate had a deletion of 2 amino acid residues at the carboxy terminus. Phylogenetic analysis showed NS1 gene of the isolate belonged to A allele. The NS1 genes of the isolate and Hong Kong isolate(Sw/HK/82) belonged to the same lineage. The relationship between the isolate and those of the H3N2 subtype SIV isolated in America is relatively far, suggesting that the geographical distribution plays a significant role in the evolution of the H3N2 subtype SIV.The target gene NS1 was subcloned into the baculovirus transfer vector pFastBac after the recombinant plasmid pMD18T-NS1 and the transfer vector pFastBac being digested with EcoRâ… a nd Xhoâ… .Then ligation product was transformed into competent cells DH10Bac.Positive clones with the target gene were identified by PCR , restriction endonuclease analysis and DNA sequencing.The result indicated that the eukaryotic expression plasmids have been constructed successfully, which were named pFastBac-NS1.