| Two pairs of primers were designed based on HA genes and NA genes of the strains of the H3N2,H9N2,H1N1 subtype of influenza virus according to GenBank. Swine influenza virus H9N2 Jinan isolate (SW/SD/JT/07) as template, the HA gene and NA gene were amplified with RT-PCR, which were cloned into pMD18-T vector, sequenced and analyzed.The result is reported as follows:As a result, the ORF of the HA gene of SW/SD/JT/07(H3N2) was 1701 bp and encoded 566 amino acids. The amino acid sequence at HA0 cleavage site in SW/SD/JT/07 was PENQTR↓G, which is non-highly pathogenic. there were the seven glycosylation sites in the HA proteins of SW/SD/JT/07(H3N2). SW/SD/JT/07(H3N2) HA have the same eceptor binding sites, the 98th is Tyr,the 134th is Gly, the 136th is Ser, the 138th is Ala, the 226th is Asn.The ORF of the NA gene of SW/SD/JT/07 (H3N2)was 1413 bp and encoded 470 amino acids. The 63th, 64th and 65th amino acid residues were not found deleted in the NA protein , The nucleotide homologies of the HA genes between SW/SD/JT/07 and the reference strains were from65.2℅-81.8, the identity of the NA genes from 65.1℅-91.8℅ ,On the basis of HA gene and NA gene phylogenetic analysis, it implied that SW/SD/JT/07 (H3N2)and the avain influenza virus H3N2 subtype had the nearer inherit distance.As a result, the ORF of the HA gene of SW/SD/JT/07(H9N2) was 1701 bp and encoded 566 amino acids. The amino acid sequence at HA0 cleavage site in SW/SD/JT/07(H9N2) was R-S-S-R-G, which is non-highly pathogenic. There were the same eight glycosylation sites in the HA proteins of SW/SD/JT/07(H9N2) and the reference strains. SW/SD/JT/07(H9N2) HA have five receptor binding sites, but the 190th amino acid residue differenced between SW/SD/JT/07(H9N2) and the reference strains. The ORF of the NA gene of SW/SD/JT/07(H9N2) was 1413 bp and encoded 470 amino acids. The amino acid sequence in the NA protein matrix was very conservative among SW/SD/JT/07(H9N2) and the reference strains. There were five antigenic sites in the NA protein, the 325th amino acid residue was D in SW/SD/JT/07(H9N2)and was N in the reference strains, the 398th amino acid residue was E in SW/SD/JT/07, DC/HB/W1/04 and CK/GS/2/99 and was D or N in the other strains. The 63th, 64th and 65th amino acid residues were deleted in the NA protein of SW/SD/JT/07, CK/SH/F/98, DC/HB/W1/04, CK/GS/2/99, SW/GD/WXL/04, SW/SD/W4/03 and SW/YN/Simao2/07. The nucleotide homologies of the HA genes between SW/SD/JT/07 and the reference strains were from 82.5℅ to 98.6℅, the identity of the NA genes from 82.2℅ to 99.1%. On the basis of HA gene and NA gene phylogenetic analysis, it implied that SW/SD/JT/07 and the avain influenza virus H9N2 subtype had the nearer inherit distance.As a result, the ORF of the HA gene of SW/SD/JT/07(H1N1) was 1701 bp and encoded 566 amino acids. The amino acid sequence at HA0 cleavage site in SW/SD/JT/07(H1N1) was IPSIQSR↓G, which is non-highly pathogenic. There were eight glycosylation sites in the HA proteins which were diffrence from the reference strains,there were six sits in the HA1 proteins and two sits in the HA2 proteins, SW/SD/JT/07 (H1N1)HA have the same receptor binding sites, but the 190th amino acid residue differenced between SW/SD/JT/07(H1N1) and the reference strains. The ORF of the NA gene of SW/SD/JT/07 was 1410 bp and encoded 469 amino acids. The amino acid sequence in the NA protein matrix was very conservative among SW/SD/JT/07(H1N1) and the reference strains. There were five antigenic sites in the NA protein, the 340th amino acid residue was P in SW/SD/JT/07(H1N1) and was S in the reference strains, the 346th amino acid residue was I in SW/SD/JT/07(H1N1) and was V in the reference strains, the 366th amino acid residue was N in SW/SD/JT/07(H1N1) and was S in the reference strains; There were eight glycosylation sites in the NA proteins which were diffrence from the reference strains; On the basis of HA gene, NA gene phylogenetic analysis, it implied that SW/SD/JT/07(H1N1) and the Influenza A virus not belong to the same branch; On the basis of NA gene phylogenetic analysis, it implied that SW/SD/JT/07(H1N1) and the avain influenza virus H1N1 subtype had the nearer inherit distance.This experiment on three different subtypes of SIV HA and NA gene sequence analysis, the prevalence of SIV in order to further control the molecular epidemiological data provide.
|
PDF Full Text Request