细胞因子人MK基因的克隆及在大肠杆菌中的高效表达(简报)  

CLONING AND EFFICIENT EXPRESSION OF CYTOKINE HUMAN MK IN E. coli

作　　者：黄建[1] 林万敏[1] 罗祖玉[1] 谢毅[2] 

机构地区：[1]复旦大学生理与生物物理学系 [2]复旦大学遗传学研究所遗传工程国家重点实验室,上海200433

出　　处：《实验生物学报》2001年第2期143-146,共4页Acta Biologiae Experimentalis Sinica

摘　　要：细胞因子Midkine(简称MK)是新发现的一类肝素结合因子家族中的一员.1988年,Kadamatsu等利用差异杂交法在经维甲酸诱导分化的小鼠畸胎瘤细胞株HM-1中首先克隆到小鼠MK基因[1].For cloning the cytokine human Midkine (MK) gene, we designed by PCgene program and synthesized a pair of PCR specific primers according to the reported human MK cDNA sequence. Total cellular RNA was extracted from a human hepatoblastoma cell line HepG2, and then the target DNA fragment was obtained by RT-PCR and subcloned into plasmid pUC118. Checked with radioisotope sequencing and ABI 377A sequencer, the nu-cleotide sequence of the cloned MK cDNA was identical with the reported one. A prokaryotic expression vector, named pBV220, was used to express the MK protein effi- ciently in E. coli strain TG1 and a predicted band of 16. 5 kD in Mr by 15% SDS-PAGE was found. The expressed recombinant protein was found in insoluble aggregated form and accounted for about 31.21% of the total cellular proteins. The first 15 N-terminal amino acid sequence analysis of this protein by Edman degradation method showed that it was accordant with that predicted from the cDNA sequence. The activity of neurite outgrowth-promoting of the MK crude samples was tested with brain cells isolated from 18-day embryos of SD rat.

关 键 词：细胞因子MK 肝素结合因子 克隆 表达 

分 类 号：Q78[生物学—分子生物学]