猪流感病毒H3N2亚型济南株HA、NA、NP基因的克隆与序列分析  

Cloning and sequence analysis of HA gene,NA gene and NP gene of swine influenza virus H3N2 subtype Jinan strain

作　　者：朱晓林[1] 凌宗帅[2] 邱莹[1] 谢之景[1] 赵宏坤[1] 姜世金[1] 张兴晓[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]济南出入境检验检疫局,山东济南250000

出　　处：《中国兽医学报》2010年第8期1060-1063,1106,共5页Chinese Journal of Veterinary Science

基　　金：山东省优秀中青年科学家科研奖励基金项目(2008BS07017);山东出入境检验检疫局科研基金资助项目(SK03-2004)

摘　　要：根据GenBank中流感病毒H3N2亚型HA、NA、NP基因序列,分别设计扩增HA、NA、NP基因的特异性引物,用RT-PCR方法扩增猪流感病毒（SIV）H3N2济南株（SW/SD/JT/07（H3N2））HA、NA、NP基因,分别将RT-PCR产物克隆入pMD18-T载体,进行测序分析。结果显示,SW/SD/JT/07（H3N2）HA基因长度约为1 701 bp,编码566个氨基酸,与参考毒株的同源性为65.2%~81.8%;HA0切割位点序列为PENQTR↓G,属非高致病性毒株;SW/SD/JT/07（H3N2）HA蛋白有7个糖基化位点,由于碱基突变缺少了1个糖基化位点,表明其抗原性及其生物学特性发生变化。SW/SD/JT/07（H3N2）与所有参考毒株HA蛋白上的受体结合位点的氨基酸很保守,即98、134、136、138、226位分别为Tyr、Gly、Ser、Ala、Asn。SW/SD/JT/07（H3N2）NA基因长为1 413 bp,编码470个氨基酸,与参考毒株的同源性为65.1%~91.8%,NA基因系统发生分析表明SW/SD/JT/07（H3N2）与禽流感病毒H3N2亚型亲缘关系最近。SW/SD/JT/07NA（H3N2）NA蛋白与参考毒株的颈区63、64、65位置均无氨基酸缺失现象,可推测其对病毒的复制能力不会有很大影响。SW/SD/JT/07（H3N2）NP基因长为1 565 bp,编码498个氨基酸。与参考毒株的同源性为79.8%~87.8%,NP基因系统发生树分析结果表明,2002年、2004年暴发的西班牙流感同源性较高,亲缘关系较近,而与中国内地广东株同源性小,亲缘关系较远。Two pairs of primers were designed based on HA genes and NA genes and NP genes of the strains of the influenza virus H3N2 subtype in GenBank.Swine influenza virus H3N2 Jinan isolate（SW/SD/JT/07（H3N2）） as template,the HA gene,NA gene and NP gene were amplified by using RT-PCR,which were cloned into pMD18-T vector,sequenced and analyzed.As a result,the ORF of the HA gene was 1 701 bp and encoded 566 amino acids.The amino acid sequence at the HA0 cleavage site was PENQTR↓G,which is non-highly pathogenic.There were seven glycosylation sites in the HA protein with a deletion,and its antigenic and biological characteristics might change.The HA had the same receptor binding sites,the 98th amino acid residue is Tyr,the 134th is Gly,the 136th is Ser,the 138th is Ala,and the 226th is Asn.The ORF of the NA gene was 1 404 bp and encoded 468 amino acids.The 63th,64th and 65th amino acid residues were not deleted in the NA protein.The ORF of the NP gene was 1 565 bp and encoded 498 amino acids.The nucleotide homologies of the HA genes between SW/SD/JT/07（H3N2） and the reference strains were from 65.2% to 81.8%,the identity of the NA genes from 65.1% to 91.8%,and the identity of the NP genes from 79.8% to 87.8%.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.On the basis of NP gene phylogenetic analysis,SW/SD/JT/07（H3N2） and the Spain influenza viruses strains isolated in 2002 and 2004 had the nearer inherit distance,but had the farer inherit with China's Mainland Guangdong strains.

关 键 词：猪流感病毒 HA基因 NA基因 NP基因 

分 类 号：S852.65[农业科学—基础兽医学]