人微小纤溶酶原cDNA在毕赤酵母中的高效表达及活性检测  

Expression of Human Microplasminogen cDNA in Pichia pastoris and Activity Detection of Expressed Microplasminogen

作　　者：王娜[1] 何成霞 邹强 苟兴华 陈松[1] 吴昊俣 蔡心析 许天恒 WANG Na;HE Cheng-xia;ZOU Qiang;GOU Xing-hua;CHEN Song;WU Hao-yu;CAI Xin-xi;XU Tian-heng(Chengdu University,Sichuan Chengdu 610106,China)

机构地区：[1]成都大学

出　　处：《西南农业学报》2019年第12期2807-2814,共8页Southwest China Journal of Agricultural Sciences

基　　金：成都市科技惠民技术研发项目(2015-HM01-00176-SF)

摘　　要：【目的】人微小纤溶酶原是由261个氨基酸组成的肽链,位于人纤溶酶原第549~790位,含有纤溶酶原活性区域,被激活后具有纤溶活性,能够降解纤维蛋白。本文旨在优化人微小纤溶酶原cDNA在毕赤酵母中的表达条件。【方法】在NCBI中获取人微小纤溶酶原氨基酸序列,根据毕赤酵母密码子偏爱性原理,设计对应的DNA序列,然后进行人工合成。合成的基因序列经测序验证后酶切与表达载体pPICZαA连接形成重组表达质粒pPICZαA-mPlg,转入大肠杆菌JM109中扩增质粒,提取阳性菌落中的重组质粒,双酶切获得人微小纤溶酶原的表达单元,然后构建4拷贝(表达单元)pPICZαA-mPlg,单酶切线性化质粒。然后电转化导入感受态毕赤酵母SMD1168中,经表型筛选、PCR筛选阳性克隆,获得高表达人微小纤溶酶原工程菌。培养工程菌后,甲醇诱导工程菌细胞让人微小纤溶酶c DNA表达,用SDS-PAGE检查其分子量,发色底物法检测微小纤溶酶原活性,通过发酵条件优化提高人微小纤溶酶原的产量,用SP-Sepharose fast flow凝胶层析方法纯化目标蛋白。【结果】SDS-PAGE检测到符合人微小纤溶酶原分子量大小的蛋白条带,同时发色底物法检测出样品中含有纤溶活性,其水平达到90 U/mL。【结论】本文为后续进行大规模表达人微小纤溶酶原打下了基础。【Objective】Human microplasminogen is a peptide chain consisted of 261 amino acids,located in 549-790 of human plasminogen and containing plasminogen active region.After being activated,it has fibrinolytic activity to degrade fibrin.This paper aims to optimize the expression of human microplasminogen cDNA in Pichia pastoris.【Method】The amino acid sequence of human plasminogen was obtained from NCBI according to the principle of codon preference of Pichia pastoris,the corresponding DNA sequence was designed and then synthe-sized,and it was cloned into the expression vector pPICZαA to obtain one copy expression unit in pPICZαA-mPlg.The expression unit of human plasminogen was obtained by double enzyme digestion of positive expression plasmid,pPICZαA-mPlg,and final a 4-copy(expression unit)pPICZα-mplg was constructed in pPICZαA.The 4 copys expression plasmid was linearized by single enzyme digestion,and was transformed into Pichia pastoris SMD1168 by electroporation.The positive colony was screened by phenotype and PCR.The screened genetic engineering Pichia pastoris was induced with methyl alcohol,the molecular weight and activity was detected by SDS-PAGE and chromogenic substrate assay,respectively.The yield of human microplasminogen was increased by optimizing fermentation and Sp-sepharose fast flow gel chromatography was used to purify the target protein.【Result】The human microplasminogen band was observed from SDS-PAGE gel and the plasmin activity was 90 U/mL.【Conclusion】This paper paves the way for subsequent large scale expression of human microplasminogen.

关 键 词：人微小纤溶酶原 甲醇诱导 发色底物法 

分 类 号：Q786[生物学—分子生物学]