人β_2-微球蛋白在pET-22b(+)表达载体中的克隆、表达与纯化

Cloning,expression and purification of human β_2-microglobulin in pET-22b(+) vector

目的 :构建人 β2-microglobulin( β2- M)基因的原核表达载体 ,在大肠杆菌中高效表达 β2 -M蛋白质并进行纯化 ,为构建MHC 肽四聚体奠定基础。方法 :从人外周血单核细胞中提取总RNA ,采用RT-PCR技术扩增出去除信号肽部分的 β2-M基因 ,克隆入pET-2 2b( + )表达载体进行诱导表达。采用离子交换层析和凝胶过滤方法对表达产物进行纯化 ,用ELISA和West ernblot法进行免疫学鉴定。结果 :成功地构建了表达载体pET-2 2b( + )- β2-M ,对表达产物进行了纯化 ,并对表达产物进行了鉴定。结论 :获得 β2- M蛋白的高效表达 。

Objective:To construct expression vector containing β_2-M gene,further express recombinant β_2-M protein in E.coli and purify recombinant β_2-M protein.Methods:The β_2-M gene absent signal peptide was amplified by using RT-PCR.Then the β_2-M gene was cloned into pET-22b(+) vector and over expressed.The expressed protein was purified,and its activity was detected by Western blot.Results:The β_2-M protein was successfully expressed and purified.Conclusion:The over expressed recombinant β_2-M protein lays a good foundation for further constructing MHC-peptide tetramer complexes to investigate the function of CTLs.