摘要
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 hemagglutinin（HA）,and neuramidinase（NA） protein of H5N1 avian influenza virus for the preparation of antibodies and to lay a foundation for genetic engineering vaccine.Methods H5A and N1A gene of avian virus were amplified with PCR and then cloned into Pichia pastoris expression vector pPIC9k containing the AOX1 promoter and α-factor signal sequence between NotⅠand SnaBⅠsite.The recombinant plasmid pPIC9k-H5A and pPIC9k-N1A were transformed into Pichia pastoris strain GS115 with electroporation.Multi-copy recombinants were screened and fermented in flasks and inducted with 1% methanol.Sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and Western-blot were used to detect the protein expression in induced supernatant.Results Ago-Gel electrophoresis and sequence certificate indicated that H5A and N1A gene were cloned into pPIC9K successfully.SDS-PAGE showed that the gene could express product with consistent molecular weight as estimated（64KD and 52KD）.The highest volume of protein expression was observed at the 3rd and 4th day.Western-blot showed that the target protein could bind with H5 subtype AIV serum.Conclusion The HA and NA genes of avian influenza virus H5N1 were successfuly cloned and expressed,which could be useful for developing diagnose reagent or vaccine of H5N1.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2011年第1期37-39,共3页
Chinese Journal of Public Health
基金
广东省科技计划项目(2007B030702001)
关键词
流感病毒
H5N1
毕赤酵母
分泌表达
influenza virus
H5N1
Pichia pastoris
secretion expression
