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目的克隆和表达H5N1型流感病毒血凝素(HA)基因和神经氨酸酶(NA)基因,为制备抗体和亚单位疫苗打基础。方法采用PCR技术扩增H5N1型流感病毒H5A和N1A基因,克隆入含有醇氧化酶-1(AOX1)启动子和分泌信号肽序列的巴斯德毕赤酵母(Pichia pastoris)表达载体pPIC9K中,构建重组载体;经电穿孔转化酵母宿主菌GS115,筛选高拷贝重组转化子,筛选后摇瓶培养,1%甲醇诱导表达;诱导培养上清经聚丙烯酰氨凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western-blot)检测蛋白表达情况。结果电泳和测序证实成功构建酵母表达载体pPIC9K-H5A和pPIC9K-N1A,重组蛋白在毕赤酵母中可以高效表达,表达蛋白分子量大小约为64和52 kD,第3和第4天表达量最高;Western-blot检测显示,目的蛋白能与H5亚型的AIV血清结合。结论成功克隆并表达H5N1型流感病毒HA、NA基因序列,为诊断试剂和疫苗开发等进一步研究奠定基础。
Objective To clone and express the hemagglutinin (HA) gene and neuraminidase (NA) gene of H5N1 influenza virus and lay the foundation for the preparation of antibody and subunit vaccine. Methods The H5N and N1A genes of H5N1 influenza virus were amplified by PCR and cloned into the expression vector pPIC9K containing the alcohol oxidase-1 (AOX1) promoter and secreted signal peptide. Recombinant vector was transformed into E.coli host strain GS115 by electroporation. High-copy recombinant transformants were screened by shake flask culture and induced by 1% methanol. The supernatant was induced to express by SDS-PAGE and protein Western blot was used to detect the protein expression. Results The yeast expression vectors pPIC9K-H5A and pPIC9K-N1A were successfully constructed by electrophoresis and sequencing. The recombinant protein was highly expressed in Pichia pastoris. The molecular weight of the recombinant protein was about 64 and 52 kD, and reached the highest on the 3rd and 4th days. Western-blot showed that the target protein could bind to AIV serum of H5 subtype. Conclusion The successful cloning and expression of HA and NA genes of H5N1 influenza virus lay the foundation for further research on diagnostic reagents and vaccine development.