猪流感病毒H9N2亚型济南株HA、NA基因的克隆与序列分析

根据GenBank中流感病毒H9N2亚型HA基因与NA基因序列,分别设计扩增HA基因与NA基因的特异性引物,用RT-PCR方法扩增猪流感病毒H9N2济南株(SW/SD/JT/07)HA基因和NA基因,分别将RT-PCR扩增产物克隆入pMD18-T载体,进行测序分析。结果表明,SW/SD/JT/07 HA基因长度为1701bp,编码566个氨基酸,HA0切割位点序列为R-S-S-R-G,属非高致病性毒株;HA蛋白有8个糖基化位点,与参考毒株糖基化位点数一致;HA蛋白有5个受体结合位点,其中在190位氨基酸与其余参考毒株存在差异。SW/SD/JT/07 NA基因长度为1413bp,编码470个氨基酸,NA蛋白基质结合部与参考毒株一致,高度保守;NA蛋白有5个抗原位点,在325位为D其余参考毒株均为N,SW/SD/JT/07与DC/HB/W1/04,CK/GS/2/99在398位为E,其余参考毒株为D或N;SW/SD/JT/07 NA蛋白与CK/SH/F/98,DC/HB/W1/04,CK/GS/2/99,SW/GD/WXL/04,SW/SD/W4/03,SW/YN/Simao2/07一样在茎区63,64,65位发生氨基酸缺失。SW/SD/JT/07 HA基因与参考毒株的同源性82.5%～98.6%,NA基因与参考毒株的同源性82.2%～99.1%。SW/SD/JT/07 HA基因与NA基因系统发生树分析表明,SW/SD/JT/07与AIV H9N2亚型亲缘关系密切。

Cloning and sequence analysis of HA gene and NA gene of swine influenza virus H9N2 subtype Jinan strain

Two pairs of primers were designed based on HA genes and NA genes of the strains of the H9N2 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 ana-lyzed. As a result,the ORF of the HA gene of SW/SD/JT/07 was 1 701 bp and encoded 566 amino acids. The amino acid sequence at HA0 cleavage site in SW/SD/JT/07 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 and the reference strains. SW/SD/JT/07 HA has five receptor binding sites, but the 190~(th) amino acid residue showed a difference between SW/SD/JT/07 and the reference strains. The ORF of the NA gene of SW/SD/JT/07 was 1 413 bp and encoded 470 amino acids. The amino acid sequence in the NA protein matrix was very conservative among SW/SD/JT/07 and the reference strains. There were five antigenic sites in the NA protein, the 325~(th) amino acid residue was D in SW/SD/JT/07 and was N in the reference strains,the 398~(th) 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 63~(th) ,64~(th) and 65~(th) 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 a-nalysis,it implied that SW/SD/JT/07 and the avain influenza virus H9N2 subtype had a nearer inherit distance.