流感病毒H1N1血凝素基因和神经氨酸酶基因在酵母菌中的表达

Expression of the hemagglutinin and neuramidinase gene of influenza A virus H1N1 in Pichia methanolica

摘要:

在成功克隆流感病毒H1N1全长HA(Hemagglulinin，HA)、NA(Neuramidinase，NA) 基因并测序的基础上，将部分基因序列克隆到表达载体pMETA上，构建了重组表达质粒pMETA/HA(52~1 557 bp)、pMETA/NA(121~1 263 bp)，电转化真核酵母菌pMAD16，甲醇诱导表达，利用Ni2+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。SDS-PAGE显示重组蛋白在酵母菌中可以高效表达，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。成功克隆和表达了流感病毒H1N1 HA、NA基因序列，为流感病毒H1N1诊断试剂和疫苗的开发等进一步的研究提供了参考。

Abstract:

On the basis of successful cloning the full length hemagglulinin (HA) and neuramidinase (NA) gene and sequence analysis of influenza virus H1N1, part of the gene was ligated into pMETA. Expression vectors pMETA/HA (52–1 557 bp) and pMETA/NA (121–1 263 bp) were constructed and expressed in pMAD16 induced by methanol. Recombinant protein was purified through Ni2+ affinity chromatography. Western blotting and ELISA were used to determine the antigenic activity of the recombinant protein. SDS-PAGE showed that the recombinant capsid gene could be overexpressed in Pichia methanolica. ELISA and Western blotting showed that the recombinant protein had antigenicity.