人β干扰素-血清白蛋白融合蛋白在毕赤酵母中的分泌表达  

Secretory Expression of the Fusion Protein IFNβ-HSA in Pichia pastoris

作　　者：雷楗勇[1] 张莲芬[1] 杨健良[1] 金坚[1] 

机构地区：[1]江南大学生物工程学院教育部工业微生物重点实验室,无锡214036

出　　处：《中国生物工程杂志》2006年第7期13-18,共6页China Biotechnology

摘　　要：利用重叠PCR技术在体外拼接IFNB和HSA基因,将得到的融合基因插入到毕赤酵母分泌型表达载体pPIC9K中,置于启动子AOX1和α交配因子信号肽的作用下,分泌表达融合蛋白IFNβ-HSA。重组质粒pPIC9K-IFNβ-HSA经SalI线性化,电击转化毕赤酵母KM71,经G418筛选得到高拷贝转化子。PCR鉴定后,用甲醇诱导表达,SDS-PAGE和Western blot分析表达的融合蛋白IFNB-HSA,表明该蛋白分子量约为90kDa,且具有HSA的抗原性;用细胞病变抑制法测定发酵液上清中融合蛋白的干扰素活性约为640IU／ml。Overlapping PCR technology was employed to splice IFNβ and HSA genes in vitro. The spliced gene was inserted into Pichia pastoris secretory vector pPIC9K. The IFNβ-HSA gene was designed for secretory expression under the control of promoter AOX1 and Mat et signal peptide in pPIC9K. The recombinant plasmid pPIC9K/IFNβ-HSA was linearized by restriction enzyme SalI and transformed into Pichia pastoris KM71 by electroporation. The recombinant strains identified by G418 selection and confirmed by PCR analysis were induced by methanol to express fusion protein IFNβ-HSA. SDS-PAGE and Western blot analysis of the fusion protein showed that the expressed fusion protein IFNβ-HSA with an apparent 90kDa molecular weight had the antigenicity of HSA. The specific activity of culture supematant was about 640IU/ml assayed by the standard atuviral activity test on WISH cells challenged with VSV virus.

关 键 词：Β干扰素 巴斯德毕赤酵母 融合蛋白 

分 类 号：Q786[生物学—分子生物学]