鼠尾胶原蛋白提取、分离、纯化方法的建立及鉴定

目的建立一种高效提取、分离、纯化鼠尾胶原蛋白的方法。方法通过对鼠尾进行剥离获得鼠尾腱,用Tris-HCl缓冲液、胃蛋白酶处理获得鼠尾胶原蛋白原液、反复使用氯化钠溶液进行分级盐析、醋酸溶液复溶进行鼠尾胶原蛋白的纯化。超纯水透析除去无机盐类获得纯化的鼠尾胶原蛋白。通过SDS-PAGE蛋白质电泳、氨基酸含量分析等技术手段鉴定。结果本研究建立的方法可以获得高纯度的鼠尾胶原蛋白,纯度达到电泳纯。与国外进口的商业化鼠尾胶原蛋白产品相比无差异。研究了提取、分离、纯化参数对得率、纯度的影响,建立了最优的鼠尾胶原蛋白提取条件,胃蛋白酶用量：1∶500,酶解时间：72 h,盐析浓度：2 mol/L,提取所用酸溶液：0.05mol/L醋酸溶液。结论为鼠尾胶原蛋白的扩大化生产提供了合适的工艺参数,为大量获得鼠尾胶原蛋白并进行更深层次的功效方面研究提供了理论支持和实践基础。

The Establishment and Appraisal of the Methods for the Extraction, Separation and Purification of Rat Tail Collagen

Objective To establish a highly efficient method for the extraction, separation and purification of rat tail collagen. Methods The rat tail tendon has been obtained through stripping the rat tail; the rat tail collagen stoste has been developed through processing with Tris-HC1 buffer, pepsin; sodium chloride solution has been repeatedly used for graded salting-out ; acetic acid has been used for the purification of collagen. To get purified rat tail collagen, inorganic salt has to be removed with ultra-pure water dialysis. Appraisal is to be made through such technical means such as SDS-PAGE protein electrophoresis, amino acid content analysis. Results Rat tail collagen of high purity can be developed with the method established in this study, whose purity can reach electrophoresis level. When compared with those commercialized rat tail collagen products imported from overseas there is no difference. The effects the parameters for the extraction, separation and purification have exerted on yield and purity has been deliberated, established The optimal conditions for the extraction of rat tail collagen has been established, pepsin usage： 1：500, enzyme solution time： 72 h, salting-out concentration： 2 mol/L, the acid solution used for extraction： O. 05 mol/L aeetum. Conclusion For rat tail collagen enlargement production to provide the proper processing parameters have been provided for the enlargement of production of rat tail collagen, for a large gain rat tail collagen and deeper efficacy research provides the theoretical support and practical basis have also been provided for the obtaining of large quantities of rat tail collagen so as to carry out deeper research on its efficacy.