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虎源流感病毒HA 基因的系统发生分析及其在杆状病毒系统中的表达

高玉伟, 夏咸柱, 侯小强, 鞠会艳, 王承宇,王铁成, 黄 耕, 邹啸环   

  1. 吉林大学畜牧兽医学院,吉林,长春
  • 出版日期:2006-06-07 发布日期:2008-07-07

The phylogenetic analysis of HA gene of tiger influenza A virus and its ex- pression in Baculovirus

GAO Yuwei,XIA Xianzhu,HOU Xiaoqiang,JU Huiyan,WANG Chengyu,WANG Tiecheng,HUANG Geng,ZOU Xiaohuan   

  • Online:2006-06-07 Published:2008-07-07

摘要: 通过对虎源流感病毒A/ Tiger/ Harbin/01/ 2003 (H5N1)的HA 基因进行克隆与序列测定,证明该基因全长为1 731 bp,读码框由1 707个碱基组成,编码568 个氨基酸。对HA 基因的进化分析表明,该基因与H5 亚型流感病毒的HA 基因同源性最高,其HA 裂解位点由6 个碱性氨基酸插入序列(RRRKKR)组成,符合高致病性禽流感病毒的分子特征。将HA 基因克隆入杆状病毒转座载体质粒pFastBacⅠ,构建重组质粒pFastBac-HA;再将该重组质粒转化DH10 Bac 感受态细菌,在体内进行重组,并经三重抗性与蓝白斑筛选,得到杆状病毒重组质粒Bacmid-HA;将Bacmid-HA 转染sf9 细胞,获得重组杆状病毒。经Western-blotting 检测,HA 蛋白在重组杆状病毒中获得表达。用感染重组病毒的sf9 细胞免疫小鼠,2 次免疫后2 周可诱导小鼠产生1∶ 8 ~1∶ 16 的血凝抑制抗体,表明虎源流感病毒的HA 基因在重组杆状病毒系统中得到了正确表达。

关键词: 虎(Panthera tigris), 流感病毒, HA 基因, 测序, 重组杆状病毒

Abstract: The HA gene of tiger influenza A virus (A/ Tiger/ Harbin/01/ 2003)was cloned and analyzed. The genetic analysis of HA gene revealed that the HA gene of the virus had a long complete ORF composed of 1070 nucleotides,coding 568 amino acids. The HA gene had a high homology with the H5 gene of influenza A virus. Moreover the HA gene displayed polybasic amino acids at cleavage site,which indicates a highly pathogenic avian influenza. The HA gene was cloned into baculovirus expression vectors pFastBacⅠ, and the recombinant plasmid of pFastBac-HA was constructed. The plasmid was transformed to DH10 Bac competent cell,and was screened by three antibiotics and blue-white patch. The bacmid-HA then was transfected into the sf 9 insect cells,and the recombinant baculovirus was obtained. The expressed products were detected by Western-blotting,and the results showed that the HA protein was expressed in recombinant baculovirus. Anti-H5N1influenza virus HI antibody (1∶8 - 1∶16) was detected in the mouse immunized with cells infected with recombinant baculovirus,which indicated that the HA recombinant protein was properly expressed.

Key words: HA gene, Influenza A virus, Recombinant baculovirus, Tiger (Panthera tigris)