双齿围沙蚕多肽的制备及其抗肺癌A549细胞活性
发布时间：2018-08-24 18:48
【摘要】：探讨双齿围沙蚕酶解多肽制备的关键技术及其对肺癌A549细胞的作用。以增殖抑制率为指标,确定最佳酶种并根据单因素试验和正交试验确定其最佳酶解工艺。经超滤获得1~3 ku的酶解液,通过阴离子色谱、凝胶过滤色谱和制备色谱对其进行进一步的分离纯化。采用四甲基偶氮唑蓝法测定沙蚕多肽PAP对肺癌A549细胞的增殖抑制率并通过倒置显微镜、吖啶橙/溴化乙锭荧光染色及Hoechst荧光染色观察其细胞形态的变化。实验结果表明最佳酶种是碱性蛋白酶,其最佳酶解条件为:酶解温度50℃、pH 11、料液比1∶1(g/mL)、酶解时间6 h、加酶量300 U/g。经纯化获得的多肽命名为PAP,其氨基酸序列为Ile-Glu-Pro-Gly-Thr-Val-Gly-Met-Met-Phe,且PAP对A549细胞的作用呈时间与剂量依赖关系,作用后细胞出现了凋亡的形态学特征。因此,PAP能明显抑制肺癌细胞A549增殖,可以诱导其发生凋亡而发挥抗肿瘤作用。
[Abstract]:To study the key technology of preparation of polypeptide by enzymatic hydrolysis and its effect on lung cancer A549 cells. The optimum enzyme species was determined by using the inhibition rate of proliferation as the index, and the optimum enzymatic hydrolysis process was determined according to the single factor experiment and orthogonal experiment. The enzymatic hydrolysis solution of 1 ~ 3 ku was obtained by ultrafiltration. It was further separated and purified by anionic chromatography, gel filtration chromatography and preparation chromatography. The proliferation inhibition rate of silkworm polypeptide PAP on lung cancer A549 cells was determined by tetramethyl azolium blue method. The morphological changes of A549 cells were observed by inverted microscope, acridine orange / ethidium bromide fluorescence staining and Hoechst fluorescence staining. The results showed that the best enzyme species was alkaline protease. The optimum conditions of enzymatic hydrolysis were as follows: hydrolysis temperature 50 鈩，
本文编号：2201713
[Abstract]:To study the key technology of preparation of polypeptide by enzymatic hydrolysis and its effect on lung cancer A549 cells. The optimum enzyme species was determined by using the inhibition rate of proliferation as the index, and the optimum enzymatic hydrolysis process was determined according to the single factor experiment and orthogonal experiment. The enzymatic hydrolysis solution of 1 ~ 3 ku was obtained by ultrafiltration. It was further separated and purified by anionic chromatography, gel filtration chromatography and preparation chromatography. The proliferation inhibition rate of silkworm polypeptide PAP on lung cancer A549 cells was determined by tetramethyl azolium blue method. The morphological changes of A549 cells were observed by inverted microscope, acridine orange / ethidium bromide fluorescence staining and Hoechst fluorescence staining. The results showed that the best enzyme species was alkaline protease. The optimum conditions of enzymatic hydrolysis were as follows: hydrolysis temperature 50 鈩，
本文编号：2201713
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