禽流感H5亚型病毒血凝素基因的表达和抗原性分析  

The expression and antigenicity analysis of the H5 subtype avian influenza HA gene

作　　者：王昱[1] 王秀荣[1] 石锐[2] 刘丽玲[1] 包红梅[1] 杨忠萍[1] 田丽娜[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所国家禽流感参考实验室兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [2]黑龙江民族职业学院,黑龙江哈尔滨150081

出　　处：《黑龙江畜牧兽医》2007年第7期23-26,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家"十五"攻关项目(2004BA519A23)

摘　　要：参考已发表的Guangdong/96/1(H5N1)基因序列设计引物,用鸡胚扩增中国农业科学院哈尔滨兽医研究所国家禽流感参考实验室保存的病毒,收集尿囊液,提取病毒RNA,经RT-PCR扩增HA基因,将PCR产物平端克隆到pMD18-T中,经酶切、PCR及测序鉴定后,定向亚克隆到pET-30a表达载体中。重组子经酶切和测序鉴定为正确后,用IPTG诱导表达,SDS聚丙烯酰胺凝胶电泳和薄层扫描分析重组蛋白的表达情况。重组蛋白r5HA经Ni-NTA His.Bind Resins纯化,并复性,用Western Blotting分析重组蛋白的抗原性。结果表明:表达产物大小约为65 ku,主要存在于包涵体中,表达量约占总蛋白的30%;同时,表达的重组蛋白具有较好的抗原性和特异性,可以用作检测H5亚型禽流感病毒抗血清的捕获抗原。The pair of primers were designed according to the reference sequence of Guangdong/96/l （ HSN1 ） . The HA gene was amplified by RT - PCR, and cloned into pMD18 -T vector. The HA gene was subeolned into pET30a vector and transformed the correct recombinant plasmid into BI21 （DE3）after checking the clone by restriction enzyme digesting, PC R, and sequencing. The recombinant protein r5 HA was purified by 6xHis Ni- NTA His Bind Resins and renatured. The antigenicity of r5HA protein was checked by western blotting. The results showed the r5HA had good antigenicity and specificity thus can be used as the capture antigen in the antibody inspection of AIV H5 subtype

关 键 词：禽流感病毒 H5亚型 血凝素 表达 抗原性分析 

分 类 号：S858.305.3[农业科学—临床兽医学]