人血管抑制因子的原核表达、纯化及活性研究

Expression and purification of recombinant human vasostatin and its activity

目的 :利用大肠杆菌表达系统表达人血管抑制因子 ,并利用镍金属螯合层析法进行纯化 ,探讨其抑制血管内皮细胞增殖的活性。方法 :采用RT PCR技术从人肝脏组织中获取人血管抑制因子的cDNA ,将其克隆至原核表达载体pQE30中进行IPTG诱导表达。表达产物经SDS PAGE分析 ,并利用镍金属螯合层析法进行纯化。3H TdR法检测纯化的血管抑制因子对血管内皮细胞增殖的抑制作用。结果 :利用pQE30表达载体表达的含 6个组氨酸尾的vasostatin蛋白 ,在SDS PAGE上表现出一条约 2 1kD的阳性条带 ,经镍金属螯合层析纯化后的蛋白经肽指纹图谱分析鉴定为目的蛋白 ,在体外可抑制人脐静脉血管内皮细胞的增殖。结论 :人血管抑制因子可在大肠杆菌中以包涵体形式高水平表达 ,并具有抑制血管内皮细胞增殖的活性。

Objective:To investigate the expression and purification of recombinant human vasostatin in E.coli as well as its activity on vascular endothelial cells.Methods:Liver total RNA was isolated from human liver and cDNA was synthesized with Kpn I and BamH I restriction sites using RT-PCR.The gene of interest was inserted into the same restriction sites in the pQE30 vector and IPTG was added to bacteria culture medium to induce the expression of vasostatin.Using metal immobilization affinity chromatography,the recombinant human vasostatin was purified.The activity of the purified protein on HUVBC proliferation was measured by[ 3H] thymidine deoxyribose uptake.Results:Recombinant human vasostatin was expressed in inclusion bodies of E.coli with a 6 histamine tag.The purity of recombinant protein was detected by SDS-PAGE as a single band at 21 kD approximately and was identified by peptide finger print analysis.The purified protein showed a dose-dependent inhibiting effect on the proliferation of human umbilical vein endothelial cells.Conclusion:Recombinant human vasostatin was expressed at a high level in inclusion bodies of E.coli and it can inhibit the proliferation of vascular endothelial cells.