广东工业大学学报 ›› 2017, Vol. 34 ›› Issue (01): 19-23.doi: 10.12052/gdutxb.160123

• 综合研究 • 上一篇    下一篇

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   

  1. 广东工业大学 轻工化工学院, 广东 广州 510006
  • 收稿日期:2016-09-28 出版日期:2017-01-09 发布日期:2017-01-09
  • 作者简介:韩雅莉(1957-),女,教授,博士生导师,主要研究方向为生化与分子生物学药物.
  • 基金资助:

    国家自然科学基金资助项目(30772739);海口市重点科技计划项目(2011-0044)

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   

  1. School of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, China
  • Received:2016-09-28 Online:2017-01-09 Published:2017-01-09

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

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℃ in a shaking incubator. TFP gene is expressed in yeast after induced with 0.5% methano and the specificity of expressed proteinidentified by SDS-PAGE. TFP fibrinolytic activity is detected by fibrin-plate method. The results showthat TFP gene has been successfully expressed in yeast Pichia pastoris, and thatthe expressed protein has fibrinolytic activity.

Key words: Tenebrio molitor L., fibrinolyric protein, gene, vector pPIC9K, Pichia pastoris GS115, expression and biological activities assay

中图分类号: 

  • R963

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