一种南极海洋芽孢杆菌抗肿瘤蛋白的分离纯化及其生物活性

发布时间：2017-12-27 03:14

本文关键词：一种南极海洋芽孢杆菌抗肿瘤蛋白的分离纯化及其生物活性　出处：《青岛科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：癌症已经成为我国人健康的第一杀手。海洋生物多样性为抗肿瘤活性物质的开发提供了一个丰富的种质资源。本课题以BEL-7402细胞为模型,从南极海水来源的微生物样本中,筛选得到了一株具有抗肿瘤活性的菌株N6-2,并鉴定该菌株为芽孢杆菌属(Bacillus)。通过超滤、硫酸铵分步沉淀、离子交换层析法从Bacillus sp.N6-2的发酵产物中分离纯化得到了一种能抑制肝癌细胞体外增殖的蛋白,命名为NCBG(a cytotoxicityβ-glucanase from Bacillus sp.N6-2)。ESI质谱确定NCBG的分子量为24.26 kDa,根据其氨基酸序列计算其分子量为24.28 kDa。采用Edman降解法测定该活性蛋白NCBG的N端15个氨基酸的序列是QTGGS FYEPF NNYNT。通过串联质谱分析得知NCBG是一种β-葡聚糖酶。从N6-2菌株的基因组中克隆得到编码NCBG的cDNA序列,该序列的ORF长729 bp,编码242个氨基酸,氨基酸序列的1-28位为信号肽,29-242位为活性蛋白,预测得到三个糖基化位点NCTW,NVSM,NRSV。预测NCBG的等电点为6.21。NCBG酶活最适pH为6.0,最适温度为40℃,且具有较好的温度稳定性与pH稳定性。NCBG对β-1,3-1,4-葡聚糖高度专一,结合克隆得到的编码序列可以推测为一种β-1,3-1,4-葡聚糖酶。NCBG对多种肿瘤细胞具有抑制作用,对A549、NCI-H460、BEL-7402、HepG2、panc-28、正常肝细胞L-02的IC50值分别为8.93、20.86、33.93、34.53、91.14、158.45μg/mL。通过侵袭与划痕实验得出NCBG能够抑制人肺癌细胞A549的迁移与侵袭。活性蛋白NCBG的分离可以为抗肿瘤新药的开发提供重要的参考。
[Abstract]:Cancer has become the first killer of people in our country. Marine biodiversity provides a rich germplasm for the development of antitumor active substances. In this study, a N6-2 strain with antitumor activity was screened from BEL-7402 samples from the Antarctic seawater samples. The strain was identified as Bacillus Bacillus. By ultrafiltration, ammonium sulfate stepwise precipitation, and ion exchange chromatography, a protein that inhibits the proliferation of hepatoma cells was obtained from the fermentation product of Bacillus sp.N6-2, named NCBG (a cytotoxicity beta -glucanase from Bacillus sp.N6-2). ESI mass spectrometry determined that the molecular weight of NCBG was 24.26 kDa, and its molecular weight was 24.28 kDa according to its amino acid sequence. The sequence of 15 amino acids at the N terminal of the active protein NCBG was determined by Edman degradation method, which was QTGGS FYEPF NNYNT. NCBG was found to be a beta glucan enzyme by tandem mass spectrometry. A cDNA sequence encoding NCBG was cloned from the genome of N6-2 strain. The ORF length of the sequence was 729 BP, encoding 242 amino acids. The 1-28 amino acid sequence was signal peptide, 29-242 was active protein, and three glycosylation sites NCTW, NVSM and NRSV were predicted. The isoelectric point of NCBG is predicted to be 6.21. The optimum pH for NCBG enzyme activity was 6, the optimum temperature was 40 C, and the temperature stability and the stability of pH were better. NCBG is highly specific to beta -1,3-1,4- dextran, which can be conjectured as a beta -1,3-1,4- glucan enzyme combined with the encoding sequence obtained from clones. NCBG has inhibitory effects on various tumor cells, and IC50 values for A549, NCI-H460, BEL-7402, HepG2, panc-28 and normal liver cell L-02 are 8.93, 20.86, 33.93, 34.53, 91.14, 158.45 g/mL respectively. NCBG can inhibit the migration and invasion of human lung cancer cell A549 by invasion and scratch test. The separation of active protein NCBG can provide important reference for the development of new antitumor drugs.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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