海蜇酶解多肽制备及其抗肿瘤活性探究.docVIP

海蜇酶解多肽制备及其抗肿瘤活性探究 　 作者：龙吟　杨永芳　甄茹　陈鹏　申林　严容芳　丁国芳 【摘要】目的　研究海蜇活性肽制备与抗肿瘤活性。方法　海蜇先经正交试验确定最佳酶解条件，然后将酶解物离心得到海蜇活性肽粗品。粗品经超滤、SephadexG-15凝胶层析、冷冻干燥纯化得到海蜇抗肿瘤活性肽，通过MTT法及Annexin V-FITC/PI双标记流式细胞测定了其对肿瘤细胞体外增值的影响。结果　碱性蛋白酶在温度为55℃，料液比为1:2，加酶量为1000 U/g，时间为4h的酶解条件下对海蜇进行酶解，酶解液中的氨基氮含量为41.3mg/100ml。从海蜇中分离纯化得到活性肽，MTT法表明：其对肿瘤细胞的增殖有较强抑制作用，且抑制作用具有明显量效依赖关系；Annexin V-FITC/PI双标记流式细胞术显示该肽可诱导肿瘤细胞凋亡。结论　经碱性蛋白酶酶解制备的活性肽对肿瘤细胞有明显的抑制作用，并能诱导肿瘤细胞凋亡。 【关键词】海蜇　酶解　抗肿瘤活性　活性肽 【Abstract】Objective To purify the anti-tumor activity peptide from jellyfish (Rhopilem aesculentum). Methods: First, the orthogonal experiment was adopted to product the crude peptide from jellyfish. Then, crude product was isolated by ultrafiltration, SephadexG-15 gel chromatography and antitumor activity peptide was obtained at last. Last, MTT method and double-labeled flow cytometry Annexin V-FITC/PI was used to determined the antitumor activity of peptide. Results Using alkaline protease at a temperature of 55℃, with the water ratio of 1:2, enzyme amount of 1000 U / g, hydrolysis time was 4h to enzyme jellyfish, and the hydrolyzate amino nitrogen contented in the product is 41.3mg/100ml. Purified from the jellyfish in a variety of active peptides, MTT method showed that its effect on tumor cell proliferation was strongly inhibited and the inhibition has a significant dose dependent, and Annexin V-FITC/PI double-labeled flow cytometry induced apoptosis of the tumor cells and apoptosis in a dose-dependent. Conclusion The peptide from jellyfish hydrolysate could significantly restrain tumor cells multiplication and induce tumor cells apoptosis. 【Key words】jellyfish enzyme anti-tumor activity active peptides 海蜇( Rhopilem aesculentum)俗称“鮓鱼”，隶属于腔肠动物门(Coelentera)、钵水母纲(Scyphozoa)、根口水母目(Rhizostomeae)、根口水母科(Rhizostomatidae)，海蜇属(Rhopilema)的无缘膜动物。海蜇的蛋白占其干重的50％，其氨基酸成分种类繁多，必须氨基酸含量丰富。海蜇等海洋生物为了适应高盐、高压、低温、缺氧等极端的海洋生活环境，其蛋白质的氨基酸组成与序列都与陆地生物蛋白有很大的不同，在种类和数量上也远远超过陆地蛋白资源。在种类繁多的海洋生物蛋白氨基酸序列中，潜在着许多具有生物活性的氨基酸序列，用特异的蛋白酶水解，就能释放出有活性的肽段。 抗肿瘤肽是一种具有抗肿瘤效果的生物