摘要
设计合成了两条编码KGDX (赖 甘 天冬 X)的寡核苷酸链 ,长度为 3 6个碱基对 ,两端分别为BamHI酶和XcoI酶切位点。将该基因插入原核高效表达载体PGEX 4T 1的tac启动子下游 ,获得重组质粒PGEX 4T 1KGDX ,转化大肠杆菌DH5a,3 7℃诱导使重组子表达。GST KGDX融合蛋白具有可溶性 ,产量为3 5mg/L培养基 ,表达量占菌体总蛋白 48 0 2 %。破碎菌体上清经谷胱甘肽 琼脂糖悬珠亲和层析法获得纯度为95 %的重组蛋白。1 2 5I7E3竞争拮抗实验结果表明 ,GST无GPIIb/IIIa结合作用 ,GST KGD融合蛋白能代替 7E3竞争性地与GPIIb/IIIa受体特异性结合 ,从而抑制血小板聚集。其IC5 0值为 40 μmol/L。
The KGDX (Lys Gly Asp X) gene was assembled from 2 synthetic oligonucleotides(36 base paris in length), using BamHI and XcoI restriction enzyme sites at the end of the gene for cloning into the expression vector PGEX4T 1 Expression of fusion proteins was directed by the tac promoter.The E.coli DH5a that contained the plasmid PGEX 4T 1 KGDX was expressed by 37℃ heat induction.The fusion protein with glutathione S transferase(GST KGDX) was purified in one step from the baterial lysate by glutathione agarose beads for affinity chromatography. GST KGDX was found to be soluble,abundant .Yields of 35mg/L of cultures was obtained. The fued GST KGDX protein was expressed in E.coli to a level of 48 02% of total celluar protein. In a competitive binding assay using 125 I 7E3 , GST KGDXinhibitedplatelet aggregation by binding with high affinity to GPIIb/III. The IC50 is 40μmol/L.
出处
《药物生物技术》
CAS
CSCD
2002年第1期16-20,共5页
Pharmaceutical Biotechnology
