猪软骨Ⅱ型胶原蛋白的提取纯化与鉴定

Study on isolation,purification and identification of collagen type Ⅱ from porcine articular cartilage

目的:从猪关节软骨中提取纯化Ⅱ型胶原蛋白,并对其进行鉴定。方法:选择猪关节软骨为提取原料,用盐酸胍去除蛋白多糖、胃蛋白酶消化、氯化钠盐析、DE22纤维树脂处理等步骤,提取纯化Ⅱ型胶原蛋白。采用SDS-PAGE、吸收光谱分析、氨基酸成分分析等方法对Ⅱ型胶原蛋白进行鉴定。结果:4 mol/L盐酸胍能有效去除蛋白多糖;分步加入胃蛋白酶的限制性酶解结果满意;氯化钠盐析浓度为2.4 mol/L时最佳。SDS-PAGE电泳结果显示提取纯化的Ⅱ型胶原蛋白与S igm a公司的产品一致。Ⅱ型胶原最大吸收峰为230 nm,氨基酸成分分析显示甘氨酸、脯氨酸和丙氨酸含量最高。结论:实验结果提示从猪关节软骨提取的Ⅱ型胶原蛋白纯度高,符合II型胶原的特征。提取材料广泛、实验条件简便,结果可靠。

Objective： To isolate and purify collagen type Ⅱ （ CⅡ ） from porcine articular cartilage and identify its purity. Methods： The porcine articular cartilage was selected as raw material. Guanidine hydrochloride was used to remove the proteoglycans. The digestion of pepsin, salting of sodium chloride, treatment with DE22 cellulose were studied for extracting C Ⅱ. The purity identification was made by SDS -PAGE, absorption spectrum and amino acid analysis. Results： The proteoglycans could be efficiently removed by 4 mol/L guanidine hydrochloride. The better results were obtained by limited enzyme digestion of pepsin added by two steps. The optimizing concentration of sodium chloride for salting C H was 2.4 mol/L.It was found that the bands of purified C H and Sigma C H were at the same location by SDS - PAGE. The absorption peak was at 230 nm. The concentrations of GLY, PRO and ALA were highest by amino acid analysis. Conclusion： The results suggest that the C H isolated from porcine articular cartilage has high purity and accords with the characteristics of collagen type Ⅱ. The improved method we adopted has significant advantages such as simple working process, convenient source of raw material and result reliability.