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血管生长抑制因子Kringle 5是目前发现的抑制血管内皮细胞增殖和肿瘤生长的活性最强的纤溶酶原片段,特异性高而毒副作用小,在肿瘤的治疗方面具有潜在的巨大价值和广阔的应用前景。根据K5基因的序列设计PCR引物,通过PCR从已有的克隆载体扩增出人纤维蛋白溶酶原的K5部分基因,将K5基因克隆入原核表达载体pET15b,经序列测定,成功构建了pET15b-K5非融合表达载体。将重组载体导入大肠杆菌中IPTG诱导表达,SDS-PAGE分析目的蛋白主要以可溶形式存在于菌体中,破碎后上清通过阳离子交换层析,纯化获得纯度大于95%的目标蛋白,脱盐后对分子量测定推测形成了三聚体。通过鸡胚绒毛尿囊膜法证明蛋白产物对鸡胚绒毛尿囊膜血管增生有一定的抑制作用。
Angiogenesis inhibitor Kringle 5 is the most active fragment of plasminogen that has been found to inhibit the proliferation and tumor growth of vascular endothelial cells. Its high specificity and low toxic side effects have great potential value in the treatment of tumors and broad Application prospects. PCR primers were designed according to the sequence of K5 gene. K5 part of human plasminogen was amplified from the existing cloning vector by PCR, and the K5 gene was cloned into the prokaryotic expression vector pET15b. The sequence of pET15b- K5 non-fusion expression vector. The recombinant vector was introduced into E. coli to induce the expression of IPTG. SDS-PAGE analysis showed that the target protein existed mainly in soluble form in the bacterial cells. The supernatant was purified by cation exchange chromatography to obtain the target protein of more than 95% purity. After desalting It is presumed that a trimer is formed on the molecular weight measurement. Chick chorioallantoic membrane method to prove that the protein product of chick chorioallantoic membrane angiogenesis have a certain degree of inhibition.