rhLEDGF p52基因原核表达载体的构建、诱导表达及蛋白纯化  

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

作　　者：赵海生[1] 王一[1] 

机构地区：[1]中国人民解放军第三军医大学附属西南医院眼科,重庆市400038

出　　处：《眼科新进展》2007年第1期25-29,共5页Recent Advances in Ophthalmology

摘　　要：目的构建rhLEDGFp52基因原核表达载体,获得rhLEDGFp52蛋白。方法利用基因重组技术将rhLEDGFp52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rhLEDGFp52进行鉴定并用Ni-NTAHis.Bind.Resin方法进行纯化。结果成功构建rhLEDGFp52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,rhLEDGFp52蛋白表达量占菌体蛋白总量的34.63%.WesternBlot结果显示rhLEDGF2蛋白能够特异性与LEDGF-ab结合。Ni-NTAHis.Bind.Resin方法进行纯化后的rhLEDGFp52,最终质量浓度达520mg·L-1,分析其纯度达87.93%.结论成功获得rhLEDGFp52蛋白,这为深入研究其生物学功能奠定了基础。Objective To construct the prokaryotic expression vector of rhLEDGF p52 gene,and obtain the protein rhLEDGF p52. Methods rhLEDGF p52 gene was constructed into the prokaryotic expression vector pET30a（ ＋ ） by DNA recombination techniques. When the constructure was confirmed correctly by enzymatic digestion and sequence analysis, rhLEDGF p52 protein was expressed by IPTG in E. coli BL21 （DE3） ,tested by Western Blot and purified by Ni-NTA His. Bind. Resin. Results The prokaryotic expression vector of rhLEDGF p52 gene was successfully constructed and the expression in soluble forms was obtained in E. coli BI21 （ DE3 ）. The quantity of expression protein accounted for 34.63% of the total bacterial protein. Western blot analysis demonstrated that rhLEDGF p52 protein could specifically integrate with LEDGF-ab. The ultimate concentration of rhLEDGF p52 protein purified by Ni-NTA His. Binck Resin was up to 520 mg·L^-1 and the purity was 87.93%. Conclusion rhLEDGF p52 protein is obtained successfully, which provides an experimental basis for the further study on its biological functions.

关 键 词：晶状体上皮 p52基因 表达 蛋白纯化 

分 类 号：R776[医药卫生—眼科]