摘要
从成人肝脏cDNA文库中 ,PCR扩增得到人vasostatin基因编码区序列 ,将此序列插入原核表达载体pQE3 0进行表达 ,SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)测定表明产物以包涵体形式存在 ,表达量占菌体总蛋白量的 5 0 %以上 .包涵体洗涤后溶于 8mol/L尿素溶液 ,在变性条件下通过镍 氨三乙酸 (Ni NTA)金属螯合亲和层析柱进行纯化后 ,再经透析进行复性 .N端氨基酸序列、分子质量、等电点等理化指标的测定结果与理论值相符 .用内皮细胞增殖试验、内皮细胞迁移试验以及鸡胚尿囊膜血管生成试验等方法进行活性检测 ,证实复性的表达产物具有抑制内皮细胞增殖和迁移。
Vasostatin gene was amplified from a human liver cDNA library by PCR method. The fragment was cloned into the pUC19 vector and sequenced. By inserting the vasostatin fragment into the pQE-30 vector, the recombinant pQE-30/vaso plasmid was constructed. After it was transformed into E.coliM15, the recombinant proteins were expressed successfully when induced with IPTG. The expressed recombinant protein accounted for more than 50% of total bacterial proteins. The expressed products formed inclusion body in E.coli. After extracted from bacterial cells and washed, it was dissolved in solution containing 8 mol/L urea and then purified by using immobilized metal ion affinity chromatography (IMAC) effectively with a purity of over 95%. Then the recombinant protein was renatured after the denaturants were removed gradually by dialysis. The protein was identified by the determination of its N-terminal amino acid sequence, molecular mass, isoelectric point etc. The results indicated that the primary structure of the expressed protein accorded with the theoretics. Endothelial cell proliferation assay, endothelial cell migration assay and chick chorioallantoic member assay, the bioactivity of vasostatin was investigated. It was proved that vasostatin can inhibit endothelial cell proliferation and migration, and inhibit angiogenesis of chick chorioallantoic member.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2003年第3期447-452,共6页
Progress In Biochemistry and Biophysics
