猪流感病毒H1N1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 H1N1 subtype Jinan strain

作　　者：朱晓林[1] 凌宗帅[2] 祝素珍[3] 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 [3]山东省出入境检验检疫局,山东青岛266000

出　　处：《中国兽医学报》2011年第1期49-54,共6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资助项目(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H1N1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)H1N1济南株(SW/SD/JT/07)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(H1N1)HA基因长为1 701bp,编码566个氨基酸;HA蛋白切割位点为IPSIQSR↓G,属于非高致病性毒株;HA蛋白有8个糖基化位点,其中有6个在HA1蛋白上,有2个在HA2蛋白上,与个别参考毒株糖基化位点个数不同;HA蛋白上的受体结合位点与多数参考毒株一致。SW/SD/JT/07(H1N1)NA基因长为1 410bp,编码469个氨基酸;NA基因颈部未发现氨基酸缺失现象;NA蛋白的头部有5个可能的抗原位点Ⅰ、Ⅱ、Ⅲ、Ⅳ和Ⅴ分别由5个区段组成,在340、346、366位分别为P、I、N,其余参考毒株在相应位置的氨基酸分别为S、V、S;NA蛋白共有8个糖基化位点,与个别参考毒株的糖基化个数不同。SW/SD/JT/07(H1N1)NP基因长为1 565bp,编码498个氨基酸,根据HA、NA、NP基因系统发生树分析,说明SW/SD/JT/07(H1N1)与2009年全球范围内暴发的H1N1甲型流感病毒的几个参考毒株亲缘关系较远,不属于同一分支;根据NA、NP基因系统发生分析表明,SW/SD/JT/07(H1N1)与H1N1亚型禽流感病毒亲缘关系最近。Two pairs of primers were designed based on HA genes, NA genes and NP genes of the strains of the HIN1 subtype of influenza virus according to GenBank. Swine influenza virus HIN1 Jinan isolate （SW/SD/JT/07） as template,the HA gene, NA gene and NP gene were amplified with RT-PCR, which were cloned into pMD18-T vector,sequenced and analyzed. As a result,the ORF of the HA gene of SW/SD/JT/07（H1N1） was 1 701 bp and encoded 566 amino acids. The amino acid sequence at HA0 cleavage site in SW/SD/JT/07（H1N1 ） was IPSIQSR G,whieh is non-highly pathogenic. There were eight glyeosylation 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 had the same receptor binding sites, but the 190th amino acid residue differed between SW/SD/JT/07 （H1N1） and the reference strains. The ORF of the NA gene of SW/SD/JT/07 was 1 410 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/O7（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 thereference strains; There were eight glycosylation sites in the NA proteins which were diffrent from the reference strains; the ORF of the NP gene of SW/SD/JT/07（H1N1） was 1 565 bp and encoded 498 amino acids. On the basis of HA gene, NA gene and NP gene phylogenetic analysis,it implied that SW/SD/JT/07 （H1N1） and the influenza A virus do not belong to the same branch; On the basis of NA gene and NP gene phylogenetic analysis,it implied that SW/SD/JT/07（HIN1） and the avain influenza virus H1N1 subtype had the nearer inherit distance.

关 键 词：猪流感病毒 HA基因 NA基因 NP基因 克隆 序列分析 

分 类 号：S852.65[农业科学—基础兽医学]