【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。更多还原

【Abstract】 The pair of primers were designed according to the reference sequence of Guangdong/96/1(H5N1).The HA gene was amplified by RT-PCR,and cloned into pMD18-T vector.The HA gene was subcolned into pET30a vector and transformed the correct recombinant plasmid into BL21(DE3)after checking the clone by restriction enzyme digesting,PCR,and sequencing.The recombinant protein r5HA 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.更多还原