花烟草植物防御素1蛋白在毕赤酵母中的表达及纯化  

Expression of Nicotiala alata defensin 1 protein in Pichia pastoris and purification of expressed product

作　　者：杨德奕 赵懿琛 龚伟伟 李昆鹏 YANG De-yi;ZHAO Yi-chen;GONG Wei-wei;LI Kun-peng(College of Tea Science,Guizhou University,The Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region Ministry of Education,Guizhou Key Laboratory of Agricultural Biological Engineering,Guiyang 550025,Guizhou Province,China;不详)

机构地区：[1]贵州大学茶学院山地植物资源保护与种质创新省部共建教育部重点实验室贵州省农业生物工程重点实验室,贵州贵阳550025 [2]武汉金开瑞生物工程有限公司,湖北武汉430223

出　　处：《中国生物制品学杂志》2021年第5期517-521,共5页Chinese Journal of Biologicals

基　　金：贵州省高层次创新型人才培养[黔科合人才(2016)4003号];中国烟草总公司贵州省公司科技项目(201608)。

摘　　要：目的在毕赤酵母中表达花烟草植物防御素1(Nicotiala alata defensin 1,NaD1)蛋白,并进行纯化。方法重组表达质粒pPIC9K-NaD1经SacⅠ内切酶线性化后,电转化至感受态毕赤酵母GS115,采用甲醇进行小量诱导表达,选择表达量相对较高的单菌落进行扩大培养,获得大量重组蛋白NaD1,经Ni-NTA亲和层析纯化后,对纯化产物进行15%SDS-PAGE分析及Western blot检测。结果纯化产物相对分子质量约20000,可与小鼠抗NaD1单克隆抗体发生特异性结合,于相对分子质量约20000处可见特异性结合条带,蛋白纯度达85%。结论在毕赤酵母中成功表达了NaD1蛋白,纯化后纯度较高,本实验为后续NaD1蛋白杀伤真菌作用机制的研究奠定了基础。Objective To express Nicotiala alata defensin 1(NaD1)protein in Pichia pastoris and purify the expressed product.Methods Recombinant plasmid pPIC9 K-NaD1 was linearized with SacⅠ,electrotransformed to competent P. pastoris GS115 and induced with methanol. The single colonies with high expression level were selected for expansion culture,and the obtained recombinant NaD1 protein was purified by Ni-NTA chromatography and identified by 15% SDS-PAGE and Western blot. Results The purified recombinant protein,with a relative molecular mass of about 20 000,showed specific binding to mouse monoclonal antibody against NaD1. A specific band with relative molecular mass of about 20 000 was observed,of which the purity reached 85%. Conclusion NaD1 protein was successfully expressed in P. pastoris,which reached a high purity after purification. This study laid a foundation of further research on the mechanism of NaD1 protein in killing fungi.

关 键 词：植物防御素1 毕赤酵母 花烟草 

分 类 号：Q512[生物学—生物化学]