摘要
根据人纤溶酶原的cDNA序列 ,利用PCR技术获得人纤溶酶原kringle 5结构域的基因 ,并将其克隆至表达质粒 pET2 5b(+)中 .重组载体 pET2 5b(+) /kringle 5转化至大肠杆菌BL2 1(DE3)中 ,经IPTG诱导表达 ,在 12kD处可见明显的表达条带 ,表达产物大部分以无活性的包涵体形式存在 .包涵体经过体外变复性 ,SP SepharoseFF离子交换柱一步纯化 ,15%SDS PAGE鉴定考染一条带 ,纯度达 95%以上 .所得到的目标蛋白能明显的抑制bFGF诱导的牛毛细血管内皮细胞增生 .
Angiostatin is a potent angiogenesis inhibitor which has been identified as an internal fragment of plasminogen that includes its first four kringle modules. The kringle 5 domain of human plasminogen would appear to be more potent than angiostatin on inhibition of basic fibroblast growth factor stimulated capillary endothelial cell proliferation. The gene encoding for kringle 5 domain of human plasminogen was obtained by PCR using human plasminogen cDNA as template. The amplified fragment was cloned into the vector pET25b(+) to construct the recombinant expression vector. Upon induction with IPTG, the E.coli BL21(DE3) containing the recombinant plasmid could express a distinct band with a molecular weight of 12?kD. Most of the kringle 5 was expressed in the form of the inclusion body without biological activity. The inclusion body was refolded in vitro and purified with SP Sepharose FF ion exchange chromatography. After single step elution, the sample was purified and it showed one band by 15% SDS PAGE analysis, which was detected by Coomassie brilliant blue stained. The purity of protein is more than 95%. The target protein also showed high activity of inhibition to bovine capillary endothelial cell proliferation which was induced by bFGF.
出处
《南京大学学报（自然科学版）》
CAS
CSCD
北大核心
2001年第2期218-222,共5页
Journal of Nanjing University（Natural Science）
基金
国家自然科学基金! ( 3 9970 1 73 )
