昆虫细胞中共表达人糖基转移酶对重组蛋白SEAP表达的影响  

The Effect of Human Glycosyltransferases Co-expression on the Expression of SEAP in Insect Cells

作　　者：唐巍玲 田苗苗[1] 黎健蓓 钟江[1] 

机构地区：[1]复旦大学生命科学学院微生物学与微生物工程系,上海200438

出　　处：《复旦学报（自然科学版）》2016年第2期232-238,共7页Journal of Fudan University：Natural Science

基　　金：国家科技重大专项(2011ZX09506-001)

摘　　要：杆状病毒-昆虫细胞表达系统有表达量高及蛋白翻译后修饰较完善等特点,但与哺乳动物相比其缺乏几种关键的糖基转移酶.许多研究表明将哺乳动物的糖基转移酶基因转入昆虫细胞中,能使得昆虫细胞表达蛋白的修饰得到有效改善.本研究通过RT-PCR克隆了人Ⅱ型N-乙酰葡萄糖胺转移酶(β-1,2-N-acetylglucosaminyltransferaseⅡ)和Ⅰ型β-1,4-半乳糖基转移酶(β-1,4-galactosyltransferase)基因,并将它们分别插入重组杆状病毒上,获得了表达GnT2和β4GalT1的重组杆状病毒.以具有糖基化修饰位点的人分泌型碱性磷酸酶(Secreted Alkaline Phosphatase,SEAP)为对象,将表达糖基转移酶的重组杆状病毒和表达SEAP的重组杆状病毒共感染细胞,Western blot分析结果表明共感染时所表达的SEAP蛋白条带分子量明显大于其单独表达时的分子量.结果表明重组病毒共感染对杆状病毒-昆虫细胞表达的SEAP有修饰作用.The baculovirus-insect cell expression system has the characteristics of high expression level and good post-translational protein modificaiton. But comparing to mammalian cells, it lacks several key glyco- syhransferases. In the current work, human β-1, 2-N-acetylglucosaminyhransferase Ⅱ （GnT2） and 13-1,4- galactosyltransferase （β4GAIT1） were obtained by RT-PCR and used to construct two baculoviruses expressing GnT2 and β4GAIT1. Then, the two recombinant viruses were used to infect insect cells together with another recombinant baculovirus expressing a human glycoprotein SEAP （secreted embryonic alkline phosphatase） to test their effect on SEAP glycosylation. Western blot showed that co-infection increased the molecular weight of SEAP. This result was confirmed by MALDI-TOF MS. It is suggested that co-infection of baculoviruses expressing human glycosyhransferase could be used to modify the baculovirus-expressed SEAP in insect cells.

关 键 词：SEAP 蛋白修饰 GnT2 β4GalT1 BAC-TO-BAC 

分 类 号：Q786[生物学—分子生物学]