人胰岛素原的N-糖基化修饰及在昆虫细胞中的表达  

N-glycosylation and Expression of Human Proinsulin in Insect Cells

作　　者：车凤玉[1] 刘子瑜[1] 张业顺[2] 张国政[2] 韦亚东[2] 

机构地区：[1]江苏科技大学生物与化学工程学院,江苏镇江212018 [2]中国农业科学院蚕业研究所,江苏镇江212018

出　　处：《蚕业科学》2013年第3期522-526,共5页ACTA SERICOLOGICA SINICA

摘　　要：人胰岛素为非糖基化修饰的蛋白质类激素,在生物体内的半衰期非常短。利用蛋白质经N-糖基化修饰后可以增强生物活性并延长其生物半衰期的特点,对人胰岛素原基因进行DNA重组改造,在其B链的N端添加保守性的N-糖基化位点后,克隆入ph/sp质粒,并转化至E.coli AcDH10Bac细胞,获得重组杆状病毒质粒Bacmid-Ig,然后转染Sf21细胞,获得含有N-糖基化位点的人胰岛素原基因(Ig)的重组杆状病毒。用Western blotting方法在感染此重组病毒的Sf21细胞中检测到分子质量约20 kD的经过N-糖基化修饰的人胰岛素原蛋白,并且在添加衣霉素的细胞表达产物中检测到该蛋白质的分子质量变小。研究结果表明,人胰岛素原在昆虫细胞中获得了表达并且被糖基化修饰。Human insulin is a non-glycosylated protein hormone.Its in vivo biological half-life is very short.Since N-glycosylation can enhance a protein＇s biological activity and prolong its biological half-life,human proinsulin gene was reconstructed through DNA recombination,in which a conservative N-glycosylation site was added to the N-terminus of its B chain.The recombinant gene was cloned into ph/sp plasmid and transformed into E.coli AcDH10Bac to prepare recombinant baculoviral plasmid Bacmid-Ig,which was subsequently used to transfect Sf21 cells to obtain the recombinant baculovirus carrying human proinsulin gene（Ig） with N-glycosylation sites.Western blotting displayed an N-glycosylated human proinsulin protein of approximately 20 kD molecular mass in Sf21 cells infected with recombinant virus.Moreover,a reduced molecular mass was detected in cellular expression product after adding tunicamycin.These research results showed that human proinsulin gene had been expressed and glycosylated successfully in insect cells.

关 键 词：人胰岛素原 昆虫细胞 N-糖基化修饰 重组杆状病毒 

分 类 号：Q786[生物学—分子生物学]