两种β2m重组质粒的构建及其表达与纯化  

Cloning and expressing of two β2m recombinant plasmids in different parts of BL21(DE3) strain of E. coli bacteria

作　　者：郭薇[1] 张建琼[2] 沈传来[2] 孟凡岩[2] 

机构地区：[1]中国药科大学生命科学与技术学院分子生物学教研室,南京210009 [2]东南大学遗传学研究中心,南京210009

出　　处：《免疫学杂志》2006年第4期460-462,465,共4页Immunological Journal

摘　　要：目的获得大量有活性的人β2m重组蛋白,为构建HLA与抗原肽的复合体及HLA四聚体作准备。方法采用RTPCR克隆法构建重组质粒,于BL21(DE3)菌中原核表达后,经金属螯合亲和层析纯化,BCAProteinAssayKit测定蛋白含量,应用SDSPAGE观察重组蛋白表达情况及Westernblot分析重组抗原的活性。结果成功地构建了两种融合表达载体PET22bβ2m及PET28aβ2m,两者的表达量分别为5～10mgL,40～80mgL,纯度均为95%。结论通过比较,确定了β2m最佳表达载体系统及表达条件,为研究轻链在原核系统中表达、纯化以及构建HLA复合体,探讨免疫识别机制奠定了基础。Objective To obtain human β2m recombination protein possessing activity for selecting the optimal scheme for large scale incubation. Methods The human β2m recombination protein was expressed in BL21（DE3） strain of E. coli bacteria. The target protein was expressed by IPTG induction and purified by affinity chromatography with 6 × his tagged resin, and the expression of β2m had been determined by SDS-PAGE and Western blotting. Results Two recombinant vectors PET-22b/β2m and PET-28a/β2m were constructed successfully. Target protein was obtained and purified. The expression of β2m was determined by BCA protein assay reagent kit. Conclusion High efficient expression of β-2m-his6 fusion protein lays the foundation for research on expression and purification of L-chain in prokaryofic system, the constroction of tetrameric peptide-HLA complexes, and exploration of the mechanism of immune recognition.

关 键 词：克隆 Β2M 基因表达 纯化 

分 类 号：R346[医药卫生—基础医学]