發(fā)布時間：2017-12-27 03:14

  本文關(guān)鍵詞：一種南極海洋芽孢桿菌抗腫瘤蛋白的分離純化及其生物活性　出處：《青島科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 菌種鑒定 分離純化 基因克隆 抗腫瘤 β-葡聚糖酶

【摘要】：癌癥已經(jīng)成為我國人健康的第一殺手。海洋生物多樣性為抗腫瘤活性物質(zhì)的開發(fā)提供了一個豐富的種質(zhì)資源。本課題以BEL-7402細(xì)胞為模型,從南極海水來源的微生物樣本中,篩選得到了一株具有抗腫瘤活性的菌株N6-2,并鑒定該菌株為芽孢桿菌屬(Bacillus)。通過超濾、硫酸銨分步沉淀、離子交換層析法從Bacillus sp.N6-2的發(fā)酵產(chǎn)物中分離純化得到了一種能抑制肝癌細(xì)胞體外增殖的蛋白,命名為NCBG(a cytotoxicityβ-glucanase from Bacillus sp.N6-2)。ESI質(zhì)譜確定NCBG的分子量為24.26 kDa,根據(jù)其氨基酸序列計(jì)算其分子量為24.28 kDa。采用Edman降解法測定該活性蛋白NCBG的N端15個氨基酸的序列是QTGGS FYEPF NNYNT。通過串聯(lián)質(zhì)譜分析得知NCBG是一種β-葡聚糖酶。從N6-2菌株的基因組中克隆得到編碼NCBG的cDNA序列,該序列的ORF長729 bp,編碼242個氨基酸,氨基酸序列的1-28位為信號肽,29-242位為活性蛋白,預(yù)測得到三個糖基化位點(diǎn)NCTW,NVSM,NRSV。預(yù)測NCBG的等電點(diǎn)為6.21。NCBG酶活最適pH為6.0,最適溫度為40℃,且具有較好的溫度穩(wěn)定性與pH穩(wěn)定性。NCBG對β-1,3-1,4-葡聚糖高度專一,結(jié)合克隆得到的編碼序列可以推測為一種β-1,3-1,4-葡聚糖酶。NCBG對多種腫瘤細(xì)胞具有抑制作用,對A549、NCI-H460、BEL-7402、HepG2、panc-28、正常肝細(xì)胞L-02的IC50值分別為8.93、20.86、33.93、34.53、91.14、158.45μg/mL。通過侵襲與劃痕實(shí)驗(yàn)得出NCBG能夠抑制人肺癌細(xì)胞A549的遷移與侵襲�；钚缘鞍譔CBG的分離可以為抗腫瘤新藥的開發(fā)提供重要的參考。
[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.
【學(xué)位授予單位】：青島科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R915

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本文編號：1340010