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Alloferon-1抗生肽串联重组表达及重组Alloferon-1体外抗肿瘤活性

浙江大学学报（医学版） 2011年第5期40卷 501-507页

作者：徐小寅严杰孙琦浙江大学医学院病原生物学系浙江杭州310058 浙江医学高等专科学校浙江杭州310053

目的:构建含胰蛋白酶酶切位点的Alloferon-1抗生肽重复串联DNA片段及其原核表达系统,了解有赖氨酸尾重组Alloferon-1(rAlloferon-1)体外抗肿瘤活性。方法:根据Alloferon-1序列不含精氨酸和赖氨酸的特点,构建C端加有含胰蛋白酶酶切位点... 详细信息

目的:构建含胰蛋白酶酶切位点的Alloferon-1抗生肽重复串联DNA片段及其原核表达系统,了解有赖氨酸尾重组Alloferon-1(rAlloferon-1)体外抗肿瘤活性。方法:根据Alloferon-1序列不含精氨酸和赖氨酸的特点,构建C端加有含胰蛋白酶酶切位点赖氨酸的14×Alloferon-1抗生肽重复串联DNA片段。采用pET42a表达载体和*** BL21DE3表达宿主菌,构建Alloferon-1重复串联DNA片段原核表达系统。SDS-PAGE联合Bio-Rad凝胶图像分析系统检测目的重组产物表达量,Ni-NTA亲和层析、胰蛋白酶酶切和Sephadex G-50层析法提纯14×rAlloferon-1-K及其rAlloferon-1-K单体。BALB/c小鼠脾细胞分别与K562、KB和SGC肿瘤细胞共培养模,采用CCK-8法检测rAlloferon-1-K、化学合成Alloferon-1(cAlloferon-1)及Alloferon-1-K(cAlloferon-1-K)体外抑制肿瘤细胞生长及增殖的活性并进行比较。结果:原核表达系统*** BL21DE3pET42a-14×Alloferon-1-K在IPTG诱导下能有效表达14×rAlloferon-1-K蛋白,其产量约为细菌总蛋白的30%。0.1～10 ng/ml的rAlloferon-1-K具有明显提高小鼠脾细胞抑制K562、KB和SGC肿瘤细胞的生长及增殖(P<0.01),rAlloferon-1-K的抗肿瘤活性与cAlloferon-1及cAlloferon-1-K比较,差异无统计学意义(P>0.05)。结论:本研究成功构建了Alloferon-1抗生肽重复串联原核表达系统,有效地提高了Alloferon-1产量;而且rAlloferon-1-K仍保持原有的体外抗肿瘤活性。

关键词：重组融合蛋白质类肽类/分离和提纯抗菌药/分离和提纯抗肿瘤药/药理学重组,遗传质粒

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红树植物海杜果内生真菌Penicillium sp．中的抗菌活性成分

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高等学校化学学报 2008年第4期29卷 749-752页

作者：韩壮梅文莉崔海滨曾艳波林海鹏洪葵戴好富汕头大学海洋生物研究所汕头515063 中国热带农业科学院热带生物技术研究所海口571101

对红树植物海丰亡果内生真菌Penicillium sp。发酵液的乙酸乙酯提取物进行了柱层析分离,得到3个化合物,经MS,NMR,^1H-^1H COSY,HMQC和HMBC等鉴定,分别为4-(3-hydroxybutan-2-yl)-3,6-dimethyl-benzene-1,2-diol(1),4-(3-hydroxybutan-2-yl)-3-methyl-6-acetylbenzene-1,2-diol(2)和3,4,5-trimethyl-1,2-benzenediol(3)。其中化合物1和2为新化合物,化合物3为新的天然产物。抗菌活性测试结果表明,化合物1和3均具有抗耐甲氧西林金黄色葡萄球菌的活性,化合物2则未显示此活性。

关键词：夹竹桃科/微生物学抗菌药/分离和提纯产黄青霉/代谢

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Polyether antibiotic CP44,161

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ACTA CRYSTALLOGRAPHICA SECTION C-STRUCTURAL CHEMISTRY 2001年第1期57卷 95-96页

作者： Allen, P Brimble, MA Turner, P Univ Sydney Sch Chem Camperdown NSW 2006 Australia

The crystal structure of antibiotic CP44,161, 6-(7-{2-ethyl-2-[5-(1-hydroxymethyl)-5-methyl-2,3,4,5-tetrahydro-2-furyl]-4,10,-12-trimethyl-1,6,8-trioxadispiro[4.1.5.3]pentadec-13-en-9-yl}-4-hydroxy-3,5-dimethyl-6-oxononyl)-2-hydroxy-3-methyl-benzoic acid monohydrate, C43H66O10.H2O, has been determined by X-ray crystallography. The molecule adopts a cyclic conformation, with a centrally located water molecule contributing to the stability of the conformation through hydrogen-bonding interactions.

关键词：抗菌药/化学抗菌药/分离和提纯结晶学 X线醚类/化学醚类/分离和提纯氢键合小单孢菌科/化学模型分子分子构象吡喃类/化学链霉菌属

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Insect immunity - Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect

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JOURNAL OF BIOLOGICAL CHEMISTRY 2001年第6期276卷 4085-4092页

作者： Lamberty, M Zachary, D Lanot, R Bordereau, C Robert, A Hoffmann, JA Bulet, P CNRS Inst Biol Mol & Cellulaire Unite Propre Rech 9022 F-67084 Strasbourg France Univ Bourgogne Zool Lab CNRS Unite Mixte Rech 5548 F-21000 Dijon France

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi, Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.

关键词：氨基酸序列抗菌药/化学抗菌药/分离和提纯抗菌药/药理学抗真菌药/化学抗真菌药/分离和提纯抗真菌药/药理学碱基序列色谱法高压液相半胱氨酸/化学 DNA引物免疫组织化学等翅目/免疫学分子序列数据肽类蛋白质构象重组蛋白质类/化学重组蛋白质类/分离和提纯重组蛋白质类/药理学序列同源性氨基酸动物

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Characterization of luminal paneth cell α-defensins in mouse small intestine -: Attenuated antimicrobial activities of peptides with truncated amino termini

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第43期275卷 33969-33973页

作者： Ouellette, AJ Satchell, DP Hsieh, MM Hagen, SJ Selsted, ME Univ Calif Irvine Coll Med Dept Pathol Irvine CA 92697 USA Univ Calif Irvine Coll Med Dept Microbiol & Mol Genet Irvine CA 92697 USA Beth Israel Deaconess Med Ctr Boston MA 02115 USA

Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including alpha -defensins, termed cryptdins, Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small. intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP(-) mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides mere markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of alpha -defensins after peptide secretion.

关键词：抗菌药/分离和提纯细菌/药物作用免疫组织化学小肠/化学小鼠近交ICR 蛋白质前体/化学蛋白质前体/分离和提纯蛋白质前体/药理学构效关系动物小鼠

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Kassinatuerin-1:: A peptide with broad-spectrum antimicrobial activity isolated from the skin of the hyperoliid frog, Kassina senegalensis

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第2期268卷 433-436页

作者： Mattute, B Knoop, FC Conlon, JM Creighton Univ Sch Med Dept Biomed Sci Omaha NE 68178 USA Creighton Univ Sch Med Dept Med Microbiol & Immunol Omaha NE 68178 USA

Kassinatuerin-1 (GFMKYIGPLI(10)PHAVKAISDL(20)***2) was isolated in high yield (75 nmol/g) from an extract of the skin of a Hyperoliid frog, the African running frog Kassina senegalensis and its sequence was confirmed by total synthesis. The peptide inhibited growth of the gram-negative bacterium Escherichia coli (minimum inhibitory concentration, MIC = 4 mu M), the gram-positive bacterium Staphylococcus aureus (MIC = 8 mu M), and the yeast Candida albicans (MIC = 70 mu M). A structurally related peptide, kassinatuerin-2 (FIQYLAPLI(10)PHAVKAISDL(20)***2) was also isolated in high yield (96 nmol/g) from the extract but was devoid of antimicrobial activity against these microrganisms. Kassinatuerin-1 may be classified with other linear, cationic antimicrobial peptides that can potentially adopt an amphipathic alpha-helical conformation but it contains almost no amino acid sequence identity with previously characterized bioactive peptides from frog skin. (C) 2000 Academic Press.

关键词：氨基酸序列两栖动物蛋白质类抗菌药/化学抗菌药/分离和提纯抗菌药/药理学无尾目分子序列数据肽类/化学合成肽类/药理学蛋白质类/药理学序列同源性氨基酸皮肤/化学葡萄球菌金黄色/药物作用动物

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Human antimicrobial peptides: Analysis and application

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BIOTECHNIQUES 2000年第4期29卷 822-+页

作者： Cole, AM Ganz, T Univ Calif Los Angeles Sch Med Dept Med Div Pulm & Crit Care Los Angeles CA 90095 USA

Antimicrobial peptides are innate host defense molecules that have a direct effect on bacteria, fungi and enveloped viruses. They are found in evolutionarily diverse species ranging from prokaryotes and plants to invertebrate and vertebrate animals. Humans express several families of antimicrobial peptides in myeloid cells and on various epithelial surfaces where they are poised to defend against pathogens. Recently, antimicrobial peptides from animals and plants have sewed as templates for the design of new therapeutic antibiotics. This review provides an introduction to the biology of human antimicrobial peptides, followed by a more derailed discussion of their isolation from tissues and biological fluids, their purification by gel electrophoresis and chromatography and assays of their antimicrobial activities.

关键词：抗菌药/分离和提纯抗菌药/药理学抗微生物阳离子肽类/分离和提纯抗微生物阳离子肽类/药理学内源性抗菌多肽类物质色谱法凝胶色谱法高压液相电泳聚丙烯酰氨凝胶 α防御素/分离和提纯 α防御素/药理学 β防御素/分离和提纯 β防御素/药理学人类

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Xanthorrhizol:: A potential antibacterial agent from Curcuma xanthorrhiza against Streptococcus mutans

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PLANTA MEDICA 2000年第2期66卷 196-197页

作者： Hwang, JK Shim, JS Baek, NI Pyun, YR Yonsei Univ Dept Biomat Sci & Engn Seoul 120749 South Korea Yonsei Univ Bioprod Res Ctr Seoul 120749 South Korea Kyung Hee Univ Dept Life Sci Yongin South Korea

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Identification, characterization and purification of the lantibiotic staphylococcin T, a natural gallidermin variant

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JOURNAL OF APPLIED MICROBIOLOGY 1999年第6期87卷 856-866页

作者： Furmanek, B Kaczorowski, T Bugalski, R Bielawski, K Bogdanowicz, J Podhajska, AJ Univ Gdansk Dept Microbiol PL-80822 Gdansk Poland Med Sch Bydgoszcz Dept Microbiol Bydgoszcz Poland Univ Gdansk Fac Biotechnol PL-80952 Gdansk Poland Univ Gdansk Sch Med Gdansk Poland Univ Gdansk Dept Genet & Cytol PL-80952 Gdansk Poland

Staphylococcin T (StT), an antibacterial agent produced by a Staphylococcus cohnii T strain, was purified to homogeneity by ammonium sulphate precipitation, gel filtration, cation exchange and fast performance liquid chromatography (FPLC). The final yield was about 20%, and over a 1000-fold increase in the specific activity was obtained. Mass determination (2166 Da), amino acid sequencing (Ile-Ala-Xaa-Lys-Phe-Leu-Xaa-Xaa-Pro-Gly-Xaa-Ala-Lys-block) and DNA sequencing demonstrated that StT is identical to gallidermin, a lanthionine-containing antimicrobial peptide. StT has a broad spectrum of bactericidal activity against Gram-positive and some Gram-negative bacteria. StT appears to damage cell membrane, and as a result causes an efflux of ions and an immediate block in macromolecular synthesis. Moreover, electron microscopic observations reveal morphological changes, with a loss of ribosomes and condensation of the nucleoid DNA. These changes are followed by a dissolution of the cell contents resulting in a bacterial ghost composed of seemingly intact cell walls with remnants of the cytoplasmic membrane and internal structure. Since StT exhibits antimicrobial activity especially against the Staphylococcus species, this compound may be of use in the treatment of staphylococcal infections.

关键词：氨基酸序列抗菌药/化学抗菌药/分离和提纯抗菌药/药理学细菌素类碱基序列色谱法色谱法高压液相采暖氢离子浓度等电聚焦质谱分析法藤黄微球菌/药物作用藤黄微球菌/生长和发育藤黄微球菌/超微结构分子序列数据肽类葡萄球菌属/化学

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Isolation of chloramphenicol from whole eggs by supercritical fluid extraction with in-line collection

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JOURNAL OF AOAC INTERNATIONAL 1999年第6期82卷 1334-1339页

作者： Pensabene, JW Fiddler, W Donoghue, DJ ARS USDA Eastern Reg Res Ctr Wyndmoor PA 19038 USA US FDA Ctr Vet Med Div Anim Res Laurel MD 20708 USA

Egg consumption, at more than 65 billion per year in the United States, represents a potentially significant source of exposure to drug residues, particularly if the laying hens are treated with antimicrobial compounds or fed a diet containing medicated feed. Residues resulting from the use of chloramphenicol (CAP) is especially problematic if this compound is not used in accordance with national registration, e.g., for the control of Salmonella microorganisms in poultry. The mast commonly used methods for the determination of CAP in biological samples require the use of large amounts of organic solvent. As a result, a less solvent intensive supercritical fluid extraction (SFE) method was developed for CAP in whole chicken eggs, and the results were compared with those for a solvent extraction procedure. In the SFE method, the egg sample is extracted with supercritical CO2 (without a modifier) at 10 000 psi (680 bar), 80 degrees C, and an expanded gas flow rate of 3.0 L/min to a total volume of 150 L. The CAP is trapped in-line on a Florisil sorbent bed. The CAP is eluted post-SFE by using the liquid chromatographic mobile phase solvent (water-methanol), and determined on a Cs column with ultraviolet detection at 280 nm. Recovery from eggs fortified at the 10 ppb level (n = 6) was 81.2 +/- 4.3%. To obtain eggs containing incurred CAP, hens were given a single daily dose of 75 mg CAP (orally by gelatin capsule) for 2 consecutive days, and the eggs were collected over a 12-day period. The mean value for "normally incurred" CAP in the eggs (n = 17) analyzed by SFE ranged from none detected to 174.5 ppb, with an overall mean of 60.5 ppb, compared with a mean of 60.4 ppb for the solvent extraction method. No significant difference in resuits was found between methods. However, the SFE method is more rapid, uses less solvent, and gives recoveries similar to those for the solvent extraction method, making it ideal for regulatory monitoring.

关键词：抗菌药/分离和提纯鸡氯霉素/分离和提纯色谱法液相/方法药物残留物/分离和提纯蛋类/分析指示剂和试剂溶剂动物女(雌)性

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