黄粉虫纤溶活性基因的克隆及在毕赤酵母中的表达及活性检测.PDF

第 34 卷 第 1 期 广东工业大学学报 Vol. 34 No. 1 2017 年 1 月 Journal of Guangdong University of Technology January 2017 doi: 10.12052/gdutxb.160123 黄粉虫纤溶活性基因的克隆及在毕赤酵母中的表达 及活性检测 韩雅莉，林非凡，林泽飞，段雪娟，朱晓琳 （广东工业大学 轻工化工学院， 广东 广州 510006 ） 摘要: 采用PCR技术, 以含有TFP(Tenebrio Fibrinolyric Proteins, TFP) cDNA质粒作为模板, 扩增出TFP基因片段. 将克隆 获得的目的基因插入到毕赤酵母表达载体pPIC9K 中, 经测序无误后, 获得重组表达质粒pPIC9K-TFP. 将重组表达质粒 线性化后电击转化到毕赤酵母GS115 中, 经筛选并PCR验证获得多拷贝整合型酵母工程菌, 对工程菌进行发酵和甲醇 诱导表达. 阳性样品经纤溶平板实验验证, 表明该表达蛋白具有生物活性. 关键词: 黄粉虫；纤溶酶；基因；载体pPIC9K；毕赤酵母； 表达及活性检测 中图分类号: R963；Q78 文献标志码: A 文章编号: 1007–7162(2017)01–0019–05 Cloning of Tenebrio Fibrinolyric Proteins gene and Expression in Pichia pastoris and Biological Activities Assay Han Ya-li, Lin Fei-fan, Lin Ze-fei, Duan Xue-juan, Zhu Xiao-ling (School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China) Abstract: In this paper, TFP fragment was firstly obtained by PCR amplification using a plasmid which contains TFP cDNA as a template. The amplified fragment wascloned into P. Pastoris expressing vector pPIC9K. After confirming through sequencing, a recombinant expression plasmid pPIC9K-TFPisobtained. The plasmid islinearized and then transformed into P. Pastoris cell GS115 by electroporation. The positive recombinantis screened and confirmed by PCR. A single colony is used fermented which grew at 30