可溶性Ⅱ型胶原蛋白提取纯化条件的研究  

Study on Optimizing Preparative Procedure for Isolating and Purifying the Soluble Collagen Type Ⅱ

作　　者：姜旭淦[1] 陈盛霞[1] 王卉放[1] 吴亮[1] 许化溪[1] 

机构地区：[1]江苏大学医学技术学院,江苏镇江212001

出　　处：《临床检验杂志》2006年第6期418-421,共4页Chinese Journal of Clinical Laboratory Science

摘　　要：目的优化实验条件,建立提取纯化可溶性Ⅱ型胶原蛋白(solublecollagentypeⅡ,SCⅡ)的可靠方法。方法选择鸡胸箭突软骨为提取原料,用盐酸胍去除蛋白多糖,对胃蛋白酶的消化方式、氯化钠盐析浓度、DEAE阴离子树脂种类进行研究。采用SDS-PAGE、吸收光谱分析、氨基酸成分分析对SCⅡ进行鉴定。结果4mol/L盐酸胍能有效去除蛋白多糖,分二步加入胃蛋白酶的消化结果满意,氯化钠盐析浓度为2.4mol/L时最佳。SDS-PAGE电泳结果显示提取纯化的SCⅡ与Sigma公司的产品一致,SCⅡ最大吸收峰为230nm,在检出的15种氨基酸中甘氨酸、脯氨酸和丙氨酸含量最高。结论改进后的方法具有材料广泛、实验条件简便、结果可靠等优点,适用于科研、临床各种规模SCⅡ的提取。所得产品纯度高、符合II型胶原的特征。Objectives To establish a reliable method for isolating and purifying soluble collagen type Ⅱ （ SC Ⅱ ） by optimizing prepara- tive procedure. Method The chicken sternal cartilage was selected as raw material. Guanidine hydrochloride was used to remove the proteoglycans. The digestion manners of pepsin, sodium chloride concentrations for salting, types of DEAE anion resin were studied for extracting SC Ⅱ, The SC Ⅱ identification was made by SDS-PAGE, absorption spectrum and amino acid analysis. Result It was convenient for pre-treatments of chicken sternal cartilage. The proteoglycans could be efficiently removed by 4 mol/L guanidine hydrochloride. The satisfied results were obtained by limited enzyme digestion of pepsin added by two steps. The optimizing concentration of sodium chloride for salting was 2.4mol/L, SDS-PAGE maps revealed that the bands of purified SC Ⅱ and standard C Ⅱ were at the same location. The absorption peak of SCⅡ was at 230nm. The concentrations of Gly ,Pro and Ala were the highest in 15 amino acids. Conclusion The improved method has significant advantages of simple working process, result reliability and convenient source of raw material, It is suitable for purifying the SC Ⅱ at variable scales in research works and clinic application. The SC iI product obtained has high purity and accords with the characteristics of collagen type Ⅱ.

关 键 词：Ⅱ型胶原蛋白 类风湿性关节炎 软骨 蛋白多糖 

分 类 号：Q512.6[生物学—生物化学]