Abstract: Objective To clone，express and characterize the H5 N 1 avian influenza virus hemagglutinin(HA)and neuramidinase(NA)genes．Methods Expression vectors，pMET A／HA(49一I 587 bp)and pMET A／NA(121· 1 200 bp)were constructed and transferred into the pMADl 6 yeast．Recombinant HA and NA were expressed inducibly by methanol，and purified through Ni2+affinity chromatography column．Antigeneity of the recombinant proteins were determined using Western Blotting and ELISA．Results The recombinant genes can be highly expressed(more than 95％of total proteins)in P．methanolica．SDS—PAGE showed that the products[tle dimmers，and Western Blotting and ELISA showed that the recombinant proteins have good antigencity．Conclusion The HA and NA proteins of H5 N1 avian influenza virus can be suceessfully cloned and expressed in P． methanolica．

Key words: H5N1 avian influenza virus, Hemagglutinin, Neuramidinase, Yeast expression

摘要： 目的克隆、表达和鉴定禽流感病毒HSNI血凝素基因(hemaggludnin，HA)和神经氨酸酶基因(neuramidinase，NA) 序列，为制备抗体和基因T程疫苗打下基础。方法在成功克隆禽流感病毒H5NI全长HA、NA基因并测序的基础 上。将部分基因序列克隆到表达载体pMET A上，构建了重组表达质粒pMET A／HA(49一l 587 bp)、pMET A／NA (121—1 200 bp)，电转化真核酵母菌pMADl6，甲醇诱导表达，利用Ni“亲和层析柱对重组蛋白进行纯化，并用 Western Blotting和ELISA方法检测其抗原性。结果重组蛋白在酵母菌中可以高效表达，SDS—PAGE显示蛋白表 达后形成了二聚体，蛋白纯度占总蛋白的95％以上，ELISA和Western Blotting实验证实，重组蛋白具有良好的抗原 性。结论本研究成功克隆和表达了禽流感病毒H5NI HA、NA基因序列，为禽流感病毒H5N1诊断试剂和疫苗的 开发等进一步的研究提供了依据。

关键词: 禽流感病毒H5NI, 血凝素, 神经氨酸酶, 真核酵母表达