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

doi:10.12052/gdutxb.160123

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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

综合研究 | 更新时间：2026-05-27

- 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
- Cloning of Tenebrio Fibrinolyric Proteins gene and Expression in Pichia pastoris and Biological Activities Assay
- 广东工业大学学报 2017年34卷第1期页码：19-23
- 作者机构：
- 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.
- 基金信息：
  
  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）
- DOI：10.12052/gdutxb.160123
  中图分类号： R963;Q78
- 网络首发：2017-01-09，
  
  纸质出版：2017
- 稿件说明：
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韩雅莉, 林非凡, 林泽飞, 段雪娟, 朱晓琳. 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[J]. 广东工业大学学报, 2017, 34(1): 19-23.

Han Ya-li, Lin Fei-fan, Lin Ze-fei, et al. Cloning of Tenebrio Fibrinolyric Proteins gene and Expression in Pichia pastoris and Biological Activities Assay[J]. 2017, 34(1): 19-23. DOI: 10.12052/gdutxb.160123.
韩雅莉, 林非凡, 林泽飞, 段雪娟, 朱晓琳. 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[J]. 广东工业大学学报, 2017, 34(1): 19-23. DOI： 10.12052/gdutxb.160123.

Han Ya-li, Lin Fei-fan, Lin Ze-fei, et al. Cloning of Tenebrio Fibrinolyric Proteins gene and Expression in Pichia pastoris and Biological Activities Assay[J]. 2017, 34(1): 19-23. DOI: 10.12052/gdutxb.160123. DOI：

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.

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 ha

s been successfully expressed in yeast

Pichia pastoris

and thatthe expressed protein has fibrinolytic activity.

关键词

Keywords

references

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