摘要
目的 为了获得具有DNA结合活性的人NF κBp5 0和p65蛋白 ,建立人NF κBp5 0和p65蛋白保守结构域的原核表达系统。方法 通过PCR法扩增获得人NF κBp5 0和p65蛋白保守结构域的cDNA ,后分别将其克隆到原核表达载体pET 2 2b( +)上 ,转化E .coliBL 2 1,经诱导表达及包涵体的变性和复性处理 ,获得可溶性p5 0和p65 ,同时用Western Blot鉴定NF κBp5 0和p65蛋白的表达 ,EMSA实验检测NF κBp5 0和p65蛋白的DNA结合活性。结果 构建了pET 2 2b/p5 0和pET 2 2b/p65原核表达质粒 ;p5 0、p65蛋白在大肠杆菌中的表达均为包涵体 ;变性、复性后得到了可溶性p5 0和p65蛋白 ,浓度达 2 18μg/ml和 15 8μg/ml;并证实可溶性p5 0和p65蛋白均具有DNA结合活性。 结论 成功建立了pET 2 2b/p5 0和pET 2 2b/p65原核表达系统 ,获得了具有DNA结合活性的NF κBp5
Objective To obtain soluble human NF κ B p50 and p65 proteins having DNA binding ability and to establish the prokaryotic expression system of NF κ B p50 and p65 proteins Rel homology domain (RHD). Methods The cDNA of NF κ B p50 and p65 RHD, obtained by using PCR technology, was cloned on prokaryotic expression vectors pET 22b (+) plasmids and expressed in Escherichia coli BL 21. The soluble p50 and p65 were obtained after inclusion bodies were denaturalized and renatured. Consequently, the expression of NF κ B p50 and p65 proteins was identified by Western blotting and their DNA binding bioactivity was confirmed by electrophoretic mobility shift assay (EMSA). Results pET 22b/p50 and pET 22b/p65 were correctly constructed. The p50 and p65 proteins were expressed as inclusion bodies in the precipitation, and their expression levels accounted for 30 2% and 24 2% of total bacterial proteins, respectively. Concentrations of soluble protein, obtained by denaturalizing and renaturing, were 218 μg/ml and 158 μg/ml, respectively. DNA binding bioactivity of the p50 and p65 proteins was identified. Conclusion pET 22b/p50 and pET 22b/p65 prokaryotic expression systems are successfully constructed, and NF κ B p50, p65 proteins with DNA binding ability are obtained.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2004年第10期878-881,共4页
Journal of Third Military Medical University
基金
国家重点基础研究发展规划资助项目 ("973"项目 ) (G19990 54 2 0 3)
国家自然科学基金资助项目 ( 30 0 80 0 0 9
30 2 0 0 2 70 )
全军医学科研"十五"计划面上项目 ( 0 1MB113)~~
