人晚期糖基化终产物受体胞质内段融合蛋白表达载体的构建与表达

Vector construction for the intracellular domain of human receptor for advanced glycation endproducts and expression of the fusion protein

目的构建人晚期糖基化终产物受体胞质内段(hRAGE-C)GST融合蛋白的重组原核基因表达载体并进行表达与纯化,研究其功能并鉴定与其相互作用的蛋白。方法采用PCR方法扩增hRAGE基因编码区的胞质内段,并将其重组于pGEX-KG载体中。重组质粒经PCR、酶切、序列鉴定分析后,转化大肠杆菌BL21,以异丙基β-D硫代半乳糖(IPTG)诱导产生hRAGE胞质内段的GST融合蛋白。结果PCR、酶切和测序结果表明所克隆的GST/hRAGE-C原核表达载体完全正确,继而表达、纯化获得了相对分子质量约35 000的融合蛋白(符合预期大小)。结论将hRAGE胞质内段构建于含有GST标记的载体上并获得融合蛋白的高效表达。

Objective To construct a eukaryotic expression vector for GST-tagged intracellular domain of human receptor foradvanced glycation end-products (hRAGE), and to study the function of the expressed fusion protein and identify its intera-cting proteins. Methods The coding sequence of the intracellular fragment of hRAGE was amplified by PCR and inserted intopGEX-KG vector, a general GST fusion protein expression vector. After PCR identification, endonuclease digestion and DNAsequencing, the recombinant was transformed into E.coli BL21 to achieve the expression of GST fusion protein induced byisopropyl-β-D-thiogalactoside (IPTG), followed by purification of the protein on glutathione-superflow resin. Results Therecombinant of GST/hRAGE-C was constructed and identified by PCR, endonucleases digestion and DNA sequencing. Afterprotein expression was achieved in E.coli, a molecnlar mass of 35 kD GST fusion protein was purified, whose molecular massandpuritywereassessedbysodiumdodecylsulfate-polyacrylamidegelelectrophoresis (SDS-PAGE).Conclusion Theexpression vector for intracellular domain of hRAGE has been successfully constructed and efficient expression of the fusionprotein is obtained, which can be of value for further studies.