高拷贝pPIC6HK-α-hEGF表达载体构建及其在毕赤酵母中的表达与鉴定  

Construction of High Copy pPIC6HK-α-hEGF Expression Vector and Its Expression and Identification in Pichia Pastoris

作　　者：吕彩云[1] 陈艳荣[1] 柏晓辉[1] Lv Caiyun;CHEN Yanrong;BAI Xiaohui(School of Life and Environmental Sciences,Huangshan University,Huangshan 245041,China)

机构地区：[1]黄山学院生命与环境科学学院,安徽黄山245041

出　　处：《安徽科技学院学报》2020年第4期22-29,共8页Journal of Anhui Science and Technology University

基　　金：安徽省教育厅重点项目(KJ2019A0611);安徽省教育厅自然科学研究一般项目(KJHS2017B15);安徽省自然重点项目(KJ2012A260)。

摘　　要：目的:为提高毕赤酵母对hEGF外源表达量,旨在为hEGF规模化生产及临床应用打下良好基础。方法:以构建载体pPIC6HK为基础,插入多份α-hEGF基因拷贝表达框,构建pPIC6HK-α-hEGF表达质粒,并电转入毕赤酵母后筛选高转化子,经发酵获得分泌表达目的蛋白,通过ELISA进行定量检测。结果:筛选到高拷贝转化子阳性菌株表达α-hEGF蛋白含量分别为133.61μg/mL和174.88μg/mL。结论:成功构建了高拷贝表达载体,α-hEGF在毕赤酵母GS115细胞中高效表达。Objective:In order to improve the expression of hEGF in pichia pastoris,to lay a good foundation for the large-scale production and clinical application of hEGF.Methods:Based on the constructed vector pPIC6HK,pPIC6HK-α-hEGF recombinant plasmids were constructed by inserting multiple copies ofα-HGF gene expression frame,which were electrotransferred to pichia pastoris and screened.The target protein was obtained based on high converters by fermentation secreted and expressed.It was detected by ELISA.Results:The expression ofα-hEGF protein was 133.61μg/mL and 174.88μg/mL by screening high copy inverter positive strain.Conclusion:This study successfully constructed a high-copy expression vector.α-hEGF was successfully expressed in pichia pastoris GS115 cells.

关 键 词：α-hEGF 重组毕赤菌株 构建 表达 

分 类 号：S71[农业科学—林学]