重组人Ⅱ型胶原250-270多肽的表达、纯化和鉴定

目的:表达与纯化重组人Ⅱ型胶原250-270多肽(rhCⅡ250-270),鉴定并确认rhCⅡ250-270多肽与预期的相符。方法:在E.coli BL 21中表达rhCⅡ250-270多肽,使用亲和层析法分离纯化。用限制性酶切、聚合酶链式反应(PCR)、基因序列测定、琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹等方法在基因和蛋白水平上对rhCⅡ250-270多肽进行鉴定。结果:纯化的rhCⅡ250-270多肽,大小约43 kD,纯度为89.3%。E coRⅠ和SalⅠ双酶切法和PCR法鉴定测得DNA大小约为500bp和650bp,核苷酸序列测定证实基因DNA序列与设计的完全相符。SDS-PAGE显示rhCⅡ250-270多肽目的蛋白条带的大小约为43 kD,W estern B lotting证实GST融合蛋白上存在CⅡ片段。结论:表达和纯化rhCⅡ250-270多肽与预期的相符,并发现此多肽具有较强的免疫原性。

Expression,purification and identification of recombinant human collagen type Ⅱ peptide 250-270

Objective: To investigate the expression,purification and identification of recombinant human collagen type II 250-270(rhCⅡ250-270) peptide.Methods: Fusion protein was expressed in E.coli BL21,and was isolated and purified by Glutathione Sepharose affinity chromatography.rhCⅡ250-270 was characterized on both gene and protein levels by restricted enzyme cleavage,PCR,DNA sequencing,agarose gel electrophoresis,sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE) and Western Blotting.Results: rhCⅡ250-270 was successfully expressed and purified.The relative molecular mass(Mr)of the expressed molecule was 43000 Dalton.The purity of purified fusion protein was 89.3 percent assessed by SDS-PAGE gel thinlayer scanning.The DNA fragments characterized by restricted enzyme cleavage of EcoRⅠ and SalⅠ and PCR amplification was 500bp and 650bp respectively.The sequence of the expressed gene was in accordance with our prediction by DNA sequencing.The purified rhCⅡ250-270 peptide was about 43000 Dalton by SDS-PAGE.It was proved that there were type II collagen fragments in our fusion protein by Western Blotting.Conclusion: The rhCⅡ250-270 peptide was successfully expressed in E.coli BL21,with high purification,the DNA sequence was in accordance with our prediction.It was demonstrated that the purificated peptide was of high immunogenicity.