截短型H1N1神经氨酸酶基因的表达及其抑制剂的高通量筛选

摘要

目的:利用毕赤酵母表达具有生物活性的H1N1神经氨酸酶并从发酵提取物中筛选其新型抑制剂.方法:截短型H1N1神经氨酸酶编码基因被亚克隆进质粒pPICZαA中以构建重组质粒pPICZαA-NA,线性化的质粒pPICZαA-NA经电转化导入毕赤酵母SMD1168中.随后,诱导鉴定正确的重组子分泌表达重组神经氨酸酶,进而利用该重组酶构建神经氨酸酶抑制剂的高通量筛选模型以用于发酵提取物的筛选.结果:重组菌株能够分泌表达具有生物活性的截短型H1N1神经氛酸酶,该酶受到两种已知神经氨酸酶抑制剂(奥司他韦和扎纳米韦)的显著抑制.在此基础上,筛选了20000多个发酵提取物,其中6个提取物对神经氛酸酶产生大于50%的抑制率.结论:具有生物活性的截短型神经氨酸酶可用于构建其新型抑制剂的高通量筛选模型.与从病毒中提取不同,利用毕赤酵母中制备神经氛酸酶制备的方法安全简便,这有利于促进高通量筛选神经氨酸酶新型抑制剂的研究.%AIM:To produce the HIN1 neuraminidase(NA)with catalytic activity by Pichia pastoris and screen novel inhibitors of NA from the extracts of microbial fermentation broths.METHODS:The truncated H1N1 NA coding gene was cloned into plasmid pPICZαA to construct the recombinant plasmid pPICZαA-NA,which was linearized and then electroporated into P.pastoris SMD1168.The correct transformants were induced by methanol for the expression of recombinant NA,which was then used to construct a high-throughput screening model for the screening of novel NA inhibitors from the extracts of fermentation broths.RESULTS:The recombinant P.pastoris could secrete the truncated NA of H1N1 with catalytic activity.Moreover,its activity was significantly inhibited by oseltamivir and zanamivir,two licensed inhibitors of H1N1 NA.Based on these,more than 20000 microibal fermentation extracts were screened,and 6 of them showed＞50% inhibition rate in the secondary screening experiment.CONCLUSION:The truncated NA with catalytic activity could be used to construct the high-throughput model for screening new inhibitors of NA.Compared with purification from vition,the preparation of NA is more convenient and safer from P.pastoris,which would facilitate the development of high-throughput assay for screening novel NA inhibitors.