赤魟软骨血管生成抑制因子的分离纯化
Purification of a novel angiogenesis inhibitory factor from Dasyatis akajei cartilage
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摘要: 确定了制备高纯度赤魟软骨血管生成抑制因子的纯化工艺,并通过胶原酶模型、CAM模型验证其生物学活性。正交实验确定了赤魟软骨组织抽提的最佳条件,抽提产率可达0.84%。丙酮分级沉淀对抽提产物进行粗分离,25~35%的丙酮分级产物的CAM血管生成抑制率达85%。优化离子交换层析、凝胶过滤、反相层析的条件,可进一步有效的获得高纯度的赤魟软骨血管生成抑制因子-I(DCAIF-I)。SDS-PAGE-考马斯亮蓝染色显示为一条带,分子量为62 kDa。活性验证结果表明,DCAIF-I对胶原酶具有一定的抑制活性,抑制率为36.3%,对CAM血管生成的抑制活性具有一定的剂量依赖关系。Abstract: The isolation and purification procedure for Dasyatis akajei cartilage angiogenesis inhibitory factor-I (DCAIF-I) was developed.DCAIF-I yield was improved by investigating the extractconditions, and DCAIF-I purification was achieved by combining several types of column chromatographies.The angiogenesis inhibitory bioactivity of DCAIF-I was identified by two models, such as the collagenase model and the blood vessel formation model of the chorioallantoic membrane of chicken embryo(CAM).The orthogonal experiment method was used to optimize the extract conditions, such as temperature, pH, stirring rate, and extract time.Under the optimized conditions, it was found that a high concentration of protein with the yield of 0.84% was obtained at 30℃, pH 6.2, stiring rate of 100 r/min, and extract for 72 h.The combination and parameter selection of ion exchange chromatography, gel filtration chromatography and reverse-phase high performance liquid chromatography achieved DCAIF-I purification to obtain the SDS-PAGE-purity preparation, The purifiedDCAIF-I was homogeneous as a single band of 62kDa in 12% SDS-PAGE stained with coomasie brilliant blue.The bioactivity of DCAIF-I was identified by the model of inhibitory activity of collagenase and the blood vessel formation of the CAM.The identification of collagenase model showed that the absorption value of DCAIF-I treated group was between the values of two control groups, which indicated that DCAIF-I could inhibit the dissolution of collagen protein with the inhibition rate of 36.3%.The identification of the CAM experiment indicates that the large area of blood vessels in DCAIF-I treated group exhibited heavy loss of color, accom panied with the decreased density of vessels.Quantitative analysisof CAM indicated that the blood vessels quantity of DCAIF-I treated group was decreased obviously with the increased dosage of DCAIF-I.Therefore, these findings have special importance in showing that DCAIF-I had the inhibitory activity on collagenase and the inhibitoryeffect of angiogenesis in the CAM.Moreover, the do sage of DCAIF-I correlated with the inhibitory effect of angiogenesis in the CAM.
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