论文部分内容阅读
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 ,为进一步利用亲和素在体外构建MHC-Ⅰ类分子肽四聚体奠定了基础
OBJECTIVE: To construct a prokaryotic expression vector of human β2-microglobulin (β2-M) gene and express β2-M protein in Escherichia coli for further study. Methods: Total RNA was extracted from human peripheral blood mononuclear cells. The β2-M gene was amplified by RT-PCR and cloned into pET-2bb (+) expression vector. The expressed product was purified by ion exchange chromatography and gel filtration and was identified by ELISA and West ernblot method. Results: The expression vector pET-2 2b (+) - β2-M was constructed successfully. The expressed product was purified and the expressed product was identified. Conclusion: The high expression of β2-M protein can lay a foundation for further use of avidin to construct MHC class I peptide tetramers in vitro