重组人干扰素α-2b在大肠杆菌中分泌表达  

Secretion expression of recombinate human interferon α-2b by Escherichia coli

作　　者：卢晨[1] 赵辉[2] 邹文艺[2] 范清林[2] 付永标 宋礼华[1,2] 

机构地区：[1]安徽大学生命科学学院,合肥230039 [2]安徽安科生物工程(集团)股份有限公司,合肥230088

出　　处：《生物学杂志》2011年第3期58-62,共5页Journal of Biology

基　　金：国家"重大新药创制"科技重大专项(编号:2008ZX09203003)

摘　　要：人干扰素α-2b原始基因在重组原核工程菌中表达量偏低,所以我们在不改变干扰素原有氨基酸组成的前提下,根据大肠杆菌密码子偏爱性使用定向突变技术对huIFNα-2b基因进行点突变。将大肠杆菌STⅡ信号肽基因与突变后huIFNα-2b基因融合并于信号肽5′端和huIFNα-2b基因3′端引入合适的酶切位点。融合基因克隆至载体pCSE,pET-22b和pPAK4L中,此3种载体分别含有组成型启动子、T7启动子和phoA启动子。融合基因在载体pCSE中表达量很低,其中约有50%的目标蛋白能够成功实现分泌。在E.coliBL21中,pET-22b经过IPTG诱导可以实现huIFNα-2b的高表达,但STⅡ信号肽不能被有效切除。含有phoA启动子的载体pPAK4L其在E.coliW3110中可以实现huIFNα-2b较高水平的分泌表达,经过低磷诱导其表达量最高可至20μg/mL(A550)菌液,约有30%的目标蛋白质信号肽能够被成功切除并分泌到胞间质中。Because of original human interferon a-2b （huIFNot-2b） gene sequence in prokaryotic expression system always with low level of target protein, the code of huIFNa-2b gene was mutagenize according to usage in prokaryotic organism without changing the amino acid compsition. The mutated gene of huIFNa-2b was fused to the secretion signal coding sequence of Escherichia coli heat-stable enterotoxin Ⅱ（ STⅡ ）. Primers was designed for introducing fitting restriction sites to the fusion gene, then cloned into vectors pCSE, pET-22b and pPAK4L. They have constitutive expression promoter, 77 promoter and phoA promoter individually. With fusion gene, pCSE expressed huIFNα-2b protein in low yield in different hosts, nearly 50% of the protein can be secreted. Induced by IPTG, pET- 22b gets high level expression of huIFNa-2b in E. coli BL21, but the STⅡ signal can not be cutted efficiently. In low-phosphate growth media, E. coli W3110 with vector pPAK4L which contains the target gene synthesized approximately 20μg huIFNa-2b/ml A550 unit of cells, and about 30% of it can be secreted into periplasimic space.

关 键 词：人干扰素Α-2B STⅡ信号肽 分泌 大肠杆菌 W3110 

分 类 号：Q789[生物学—分子生物学]