鸭源H9N2亚型禽流感病毒血凝素基因的克隆、序列分析及原核表达

鸭源H9N2亚型禽流感病毒血凝素基因的克隆、序列分析及原核表达 Cloning,Sequence Analysis and Expression of the Hemagglutinin（HA）Gene of H9N2 Duck-Derived Avian Influenza Virus 张瑞华 ZHANG Rui-hua 1 2 first-author 金梅林 JIN Mei-lin 1 2 王贵华 WANG Gui-hua 1 2 赵思婷 ZHAO Si-ting 1 2 喻正军 YU Zheng-jun 1 2 陈焕春 CHEN Huan-chun 1 2 华中农业大学动物医学院 华中农业大学动物医学院 College of Animal Science and Veterinary Medicine,Huazhong Agricultural University College of Animal Science and Veterinary Medicine,Huazhong Agricultural University 华中农业大学动物医学院,湖北,武汉,430070 华中农业大学动物医学院,湖北,武汉,430070 参照GenBank收录的H9N2亚型禽流感病毒血凝素（HA）基因序列设计并合成引物,以鸭源H9N2亚型禽流感病毒RNA为模板,利用RT PCR方法扩增了预计约1700bp的HA基因,将此扩增产物克隆进pMD18 T载体,采用限制性酶切及序列测定鉴定阳性重组克隆子.测序结果表明HA基因长为1683bp.基于HA蛋白的信号肽在表达中的负面作用,本研究通过基因工程手段缺失HA蛋白位于起始的信号肽的编码序列,获得了缺失HA蛋白信号肽的HA基因,并将其亚克隆到pGEX KG中,与GST融合表达.SDS PAGE结果显示:融合表达的蛋白分子量约为90×103.Western印迹表明表达蛋白具有免疫活性. According to published HA gene sequence of H9N2 AIV,We designed and synthesized primers,A 1 700 bp fragment was amplified from H9N2 duck-derived Avian Influenza virus RNA by RT-PCR and cloned into the pMD18-T vector.The recombinant plasmid was proved to be true by enzyme analyis and sequencing.The sequence analysis showed that HA gene consisted of 1683bp. Based on the negative function of signal peptide of HA gene in expression,we lacked the signal peptide of HA gene which lies in initial coding sequence through the genetic engineering means and obtained the HA gene which lacked signal peptide.Then partial fragment of HA was subcloned into prokaryotic expression vector pGEX-KG,and was expressed in E.coli Bl21 with the GST protein with a fusion product of 90×10 3 by SDS-PAGE analysis.Western blotting indicated that the expression protein had immunogenicity. 禽流感病毒 血凝素基因 原核表达 avian influenza virus hemagglutinin （HA）gene prokaryotic expression S852.659 国家"十五"科技攻关项目（2004BA519A07） 2004-06-01 2021-04-01 6