rhLEDGF p52基因原核表达载体的构建、诱导表达及蛋白纯化(英文)

Construction of prokaryotic expression vector of rhLEDGF p52 gene、inducing expression and purification of its protein

目的 :构建 rhLEDGF p52 基因原核表达载体 , 获得rhLEDGF p52 蛋白。方法:利用基因重组技术将 rhLEDGF p52 基因构建于原核表达载体 pET30a(+)中,经酶切、测序鉴定证实构建完全正确后,通过 IPTG 诱导在大肠杆菌 BL21(DE3)中获得表达,经免疫蛋白印迹试验对 rhLEDGF p52 进行鉴定并用 Ni-NTA His.Bind.Resin 方法进行纯化。结果:成功构建 rhLEDGF p52 基因原核表达载体,在大肠杆菌 BL21(DE3)中获得可溶性形式表达,rhLEDGF p52蛋白表达量占菌体蛋白总量的 34.63%。Western Blot 结果显示 rhLEDGF2 蛋白能够特异性与 LEDGF-ab 结合。Ni-NTA His.Bind.Resin 方法进行纯化后的 rhLEDGF p52,终浓度达 520mg/L,分析其纯度达 87.93%。结论:获得 rhLEDGF p52 蛋白,这为深入研究其生物学功能奠定了基础。

AIM： To construct the prokaryotic expression vector of rhLEDGFp52 gene,to obtain the rhLEDGF p52 protein. METHODS.. rhLEDGFp52 gene was constructed into a prokaryotic expression vector pET30a （＋） by recombinant DNA techniques and was identified by enzymatic digestion and sequence analysis, rhLEDGFp52 protein was induced expression by IPTG in E.coli BL21 （DE3）, it was tested by Western blot and was purified by Ni-NTA His.Bind.Resin. RESULTS.. We Successfully constructed the prokaryotic expression vector of rhLEDGFp52 gene and obtained its expression in E.coli BL21 （DE3）,it was expressed in a soluble form and detected up to 34.63% of the total bacterial protein expressed in E.coli BL21 （DE3）.Western blot analysis demonstrated that rhLEDGFp52 protein could spicifically integrate with LEDGF-ab.After purified by Ni-NTA His.Bind. Resin, the ultimate concentration of purified rhLEDGFp52 protein was 520μ g/ml and its purity was 87.93%. CONCLUSIONS： rhLEDGF p52 protein was obtained that provides an experimental basis for the further study of the biological function of rhLEDGFp52 protein.