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

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.