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.
出处
《实验生物学报》
CSCD
北大核心
2001年第2期143-146,共4页
Acta Biologiae Experimentalis Sinica
关键词
细胞因子MK
肝素结合因子
克隆
表达
Cytokine human midkine (MK). Cloning. Prokaryotic expression.