摘要
目的克隆表达血管生成抑制因子arresten基因,并探讨其生物学活性。方法利用基因重组技术,从含有人arresten基因的克隆载体pGEMArr上切下目的基因片段,亚克隆至含T7启动子的原核表达载体pRSET中,构建表达载体pRSETAN。将重组质粒pRSETAN转化入宿主菌E.coliBL21(DE3)中,用IPTG进行诱导表达。菌体经超声波破碎,镍柱亲和层析纯化并复性。采用噻唑蓝(MTT)比色法测定表达蛋白抑制血管内皮细胞的活性。结果酶切鉴定和测序验证,表达载体构建正确。在表达宿主菌中,arresten基因获得了高效表达,表达量占菌体总蛋白质的27%;表达产物经亲和层析纯化后,蛋白质纯度达96%。经复性,重组蛋白可显著抑制血管内皮细胞生长因子促脐静脉内皮细胞的增殖作用。结论人arresten基因能在pRSET表达系统中得到高效表达;复性后表达蛋白能有效抑制血管内皮细胞的增殖。
Objective To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHI and Pst I restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni~ 2+ chelation affinity chromatography and refolded.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed using the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was highly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×10~3 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could signigicantly suppress proliferation of human umbilical vein endothelial cels (HUVECs) induced by vascular endothelial growth factor (VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelial cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第5期616-617,共2页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(30271242
30371396
