沼水蛙抗菌肽brevinin-2GHa1分离纯化及结构分析

目前,已自青蛙皮肤分泌物中分离获得多种具有较强抗菌活性的多肽．本文利用电刺激法自沼水蛙背腺和耳后腺获得其皮肤分泌物,利用凝胶过滤色谱(Sephadex G-50)和反相高效液相色谱 (reverse-phase high performance liquid chromatography, RP-HPLC)分离纯化,获得一种新型抗菌肽,命名为brevinin- 2GHa1. 抑菌实验显示,该抗菌肽对革兰氏阳性菌和革兰氏阴性菌均有抑制作用,对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和沙门氏菌的最小抑制浓度分别为： 7.8、3.9、2.0 μg/mL和250.0 μg/mL. 该抗菌肽在水中为无规卷曲结构,在浓度为10 mmol/L SDS水溶液和不同浓度三氟乙醇水溶液中则呈α-螺旋结构,该抗菌肽结构的研究对阐明其抑菌机制具有重要作用.     

中国林蛙皮肤抗菌肽抗菌的特性

从林蛙皮肤中分离到具有抗菌活性的多肽混合物——多肽FⅢ。抑菌实验表明,林蛙皮肤中小分子活性肽对革兰氏阳性细菌、革兰氏阴性细菌都具有一定的抗菌作用,并且此粗提物的抗菌活性远远高于传统食品防腐剂苯甲酸钠和山梨酸钾的抗菌活性。     

解淀粉芽孢杆菌KN-BL-1及其发酵豆粕产抗菌肽类物质的研究

以牛津杯法比较解淀粉芽孢杆菌KN-BL-1及其发酵豆粕的抑菌效果,通过酸沉淀法分离粗多肽,确定产生抑菌效果的为多肽类物质,通过DEAE-Sepharose离子交换层析及Sephadex G-25凝胶过滤层析分离纯化粗多肽以研究其各组分的抑菌效果,并使用4000 Q TRAP液相色谱-质谱联用仪初步确定抗菌肽样品的分子量范围.结果表明,KN-BL-1及其发酵豆粕浸提液对S.aureus等革兰氏阳性菌均有明显的抑菌效果;粗多肽经过层析分离纯化后,三个峰表现抑菌特性,其中有一个抑菌效果最好,抑菌圈清晰,直径为19.8 mm.     

黑眶蟾蜍皮肤抗菌肽的制备及其性质

收集黑眶蟾蜍皮肤分泌物,经Sephadex G-25去除大分子蛋白后,利用微量测定法进行抗菌活性分析。结果发现:黑眶蟾蜍皮肤分泌物对革兰氏阳性菌——金黄色葡萄球菌、枯草芽孢杆菌的抑制作用较强,对革兰氏阴性菌中的嗜水气单细胞菌也表现出较强的抑制作用,对溶藻弧菌、副溶血弧菌、河流弧菌、大肠杆菌的抑制相对较弱。利用胰蛋白酶对黑眶蟾蜍皮肤抗菌肽水解后,其抗菌活性消失。将黑眶蟾蜍皮肤抗菌肽在37~95℃和pH 2.5~5.0下保温,发现其抗菌活性成分对热及酸耐受性较强。黑眶蟾蜍皮肤分泌物在低浓度无溶血活性。     

家蝇幼虫抗菌肽的超声诱导及分离纯化和活性研究

目的:分离纯化家蝇(Musca domestica)幼虫免疫血淋巴中的抗菌肽,研究抗菌肽的性质和抑菌特性.为进一步研究家蝇幼虫抗菌肽的产生及应用提供实验基础.方法:通过超声处理诱导家蝇幼虫产生免疫血淋巴,经沸水浴热变性,结合高速离心,两步CM-Sepharose离子交换层析,冷冻干燥浓缩等步骤,分离得到一种具抗菌活性的蛋白质.用Tricine-SDS-PAGE鉴定抗菌肽的纯度和性质,抑菌试验分析抗菌肽的抑菌特性.结果:300W,50Hz超声处理60s能够诱导家蝇幼虫产生抗菌肽,经分离纯化后Tricine-SDS-PAGE凝胶电泳结果显示为一条带,相对分子量大约为5.8kD.纯化蛋白对阴沟肠杆菌,绿脓杆菌等G-杆菌的抑菌活性较强,而对金黄色葡萄球菌效果不显著.结论:诱导和纯化了一种家蝇幼虫抗菌肽,为此类活性物质的分离纯化提供了有效的方式.     

绵羊生殖道抗菌肽

以屠宰场收集的新鲜、健康、雌性绵羊生殖器官为原材料．采用乙酸浸提、透析、Sephadex G-50凝胶过滤层析和反相高效液相色谱(RP-HPLC)等方法分离纯化绵羊生殖道抗菌肽．以G+、G-和真菌为抗菌活性检测指示菌株,利用薄层琼脂糖孔穴扩散法、微量肉汤稀释法进行抗菌活性检测．对分离纯化所得纯品进行分子质量质谱测定、纯度鉴定、N端测序,并对其性质进行研究．结果表明：分离纯化所得两个绵羊生殖道抗菌肽分子质量分别为4820.47 u和4012.5 u,N端部分氨基 酸序列分别为AYVLDEPKP和YDSGA．对G+细菌(S. aureus ATCC2592、Streptococcu ATCC55121)、G-细菌(E． coli ATCC25922)、真菌(C． albicans ATCC2002)均具有良好的抑菌活性．对家兔红细胞无溶血活性,对人血液凝固无影响．目前未见有从绵羊生殖道分离纯化得到抗菌肽的报道,并且这一研究结果进一步证实抗菌肽在多种动物生殖道天然免疫防御方面起着重要作用．     

中国对虾PC-Ⅲ系列抗菌肽的分离纯化及活性

以我国主要经济海产品中国对虾（Penus chinensis）为研究对象,通过Sephadex G-50、RP-HPLC等技术分离纯化到PC-Ⅲ系列中国对虾天然抗菌肽。经初步鉴定,该系列抗菌肽对革兰氏阴性和革兰氏阳性菌都表现出程度不一的抑菌活性,且不同程度地影响小白鼠离体回肠肌收缩,但无丝氨酸蛋白酶抑制剂活性。用MALDI-TOF质谱对样品进行分析,检测到分子量分别为1071Da和1311Da的两种抗菌肽。这些抗菌肽对对虾抵御微生物的侵袭具有重要的作用。     

虎纹蛙皮肤分泌物抗氧化二肽——Gln/Lys-Met的纯化及鉴定

目前已自青蛙皮肤分泌物中提取出多种属于“第三套抗氧化系统”的多肽,它们具有较强的抗氧化作用.本文从虎纹蛙皮肤分泌物中提取具有抗氧化活性的多肽物质,并检测其活性.利用电刺激虎纹蛙背部腺和耳后腺获得其皮肤分泌物,利用凝胶过滤色谱Sephadex G-50和反相高效液相色谱（reverse-phase high performance liquid chromatography, RP-HPLC）进行分离纯化.以2,2-二苯基-1-苦肼基（2,2-diphenyl-1- picrylhydrazyl, DPPH·）清除率为指标,测定多肽的抗氧化活性,经ESI- MS（electrospray ionization mass spectrometry）质谱测定该多肽的相对分子质量.结果显示,自虎纹蛙皮肤分泌物中获得一种具有抗氧化活性的二肽,其分子质量为0.277 kD,可能的氨基酸序列为Gln/Lys-Met.虎纹蛙皮肤分泌物中的该二肽是发挥抗氧化作用的重要组成部分.     

蜘蛛抗菌肽研究进展

蜘蛛活性多肽研究主要集中于蜘蛛毒液中作用于离子通道的神经毒素多肽。但近年来,一些蜘蛛抗菌肽不断被分离纯化,其结构和抗菌活性也被广泛深入研究,这将成为蜘蛛活性多肽研究领域的一个新热点。在蜘蛛毒液和血液中,存在不同种类的抗菌肽,其多肽长度、结构、抗菌作用各不相同。而且,有些抗菌肽甚至具有抗肿瘤作用。概述了蜘蛛抗菌肽在结构和功能方面的研究进展。     

Isolation and purification of small antimicrobial peptide from larvae of mealworm, Tenebrio molitor

昆虫抗菌肽具有良好的抑菌效果,有望开发成新一代抗生素.本文以金黄色葡萄球菌和大肠杆菌混合液作为诱导源,采用针刺法使黄粉虫Tenebrio molitor L.幼虫感染微生物产生抗菌肽,并对抗菌肽进行了提取、色谱分离纯化及抑菌活性检测.结果显示,诱导组和对照组的三氟乙酸粗提物无抑菌活性;经SephadexG50、Superdex Peptide凝胶色谱分离后,从诱导组和对照组均可获得对革兰氏阳性菌金黄色葡萄球菌、枯草芽孢杆菌有抑菌作用的组分,而且诱导组活性明显高于对照组;通过Resource 15RPC反相色谱分离纯化,从诱导组获得一具有明显抑制革兰氏阳性菌的组分,质谱检测该组分为混合肽,主要由分子量为1 876.21u、1 904.21u的小肽组成,可能是一种比Thanatin分子量更低的昆虫抗菌肽.     

Purification,characterization, and sequencing of a novel type of antimicrobial peptides,Fa-AMP1 and Fa-AMP2, from seeds of buckwheat (Fagopyrum esculentum Moench.)

Novel antimicrobial peptides (AMP), designated Fa-AMP1 and Fa-AMP2, were purified from the seeds of buckwheat (Fagopyrum esculentum Moench.) by gel filtration on Sephadex G75, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They were basic peptides having isoelectric points of over 10. Fa-AMP1 and Fa-AMP2 had molecular masses of 3,879 Da and 3,906 Da on MALDI-TOF MS analysis, and their extinction coefficients in 1% aqueous solutions at 280 nm were 42.8 and 38.9, respectively. Half of all amino acid residues of Fa-AMP1 and Fa-AMP2 were cysteine and glycine, and they had continuous sequences of cysteine and glycine. The concentrations of peptides required for 50% inhibition (IC50) of the growth of plant pathogenic fungi, and Gram-positive and -negative bacteria were 11 to 36 microg/ml. The structural and antimicrobial characteristics of Fa-AMPs indicated that they are a novel type of antimicrobial peptides belonging to a plant defensin family.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

家蚕拟抗微生物肽Gloverins基因（Bmglv）的原核表达及抗菌活性鉴定

家蚕Bombyx mori基因组全测序于2004年完成后,由家蚕基因组注释发现了35条拟抗微生物肽基因序列。本研究选取其中的5条Gloverins同系物（isoforms）基因(BmglvB,BmglvA2 ,BmglvA3,BmglvA5和BmglvA6 ),克隆至pET-21d载体,转化Escherichia coli Rosetta^TM（DE3）宿主菌进行表达;克隆表达的5个家蚕Gloverins同系物基因大部分以可溶形式表达,经Ni-NTA亲和层析纯化和Sephadex G-10脱盐处理后,采用平板孔穴抑菌法测定其抗菌活性。结果表明：注释发现的5条家蚕拟抗微生物肽Gloverins同系物基因,具有与在其他昆虫中已鉴定报道的Gloverin相似的抗革兰氏阴性细菌的功能,实验确认了它们就是家蚕Gloverins抗微生物肽基因。     

Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.     

Subcellular distribution of enkephalin precursor proteins in rat dental pulp and the usefulness as a substrate for enkephalin-producing enzymes

The subcellular distribution of enkephalin (EK) precursor proteins was investigated to clarify the intracellular site of biosynthesis of EK in rat dental pulp tissue. The contents of met-EK-like peptides in nuclear, microsomal, and supernatant fractions of the pulp tissue were markedly increased after sequential digestion with trypsin and carboxypeptidase B, indicating the enrichment of the precursors in these fractions. Sephadex G-100 gel filtration showed a common peak of the precursor proteins in the homogenate and its microsomal and supernatant fractions, and the molecular weight was determined to be about 58,000 by SDS polyacrylamide gel electrophoresis. Both the partially purified precursor protein from the supernatant fraction and N alpha-benzoyl-DL-arginine-beta-naphthylamide (BANA) were used as substrates for a lysosomal enzyme separated by Sephadex G-75 gel filtration. The major peak of EK-producing activity of the enzyme was identical with that of BANA-degrading activity of the enzyme. These results demonstrate the EK-producing activity of lysosomal proteinase, and also indicate the usefulness of the two substances as substrates for the enzyme.     

Isolation and characterization of calcitonin from pericardium and esophagus of eel.

Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.     

Labeling of beta-endorphin and beta-lipotropin with iodine 125

5 micrograms of human beta-endorphin were labelled with 2 mCi 125I by the chloramine T technique. After two gel filtrations on Sephadex G-15 and on Sephadex G-50 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer was obtained with a specific activity about 150 microCi/ug. Kept at + 4 degrees C, the tracer remained utilizable for 30 days without loss of immunoreactivity. The labelling with lactoperoxydase and the use of another gel filtration method (filtration on Aca 202) gave a 125I beta-END tracer with the same immunoreactivity. The binding of this tracer to the antibody of an anti-beta-END antiserum diluted at 1/8000 was 32% with a non specific binding of 2%. 5 micrograms of human beta-lipotropin were labelled with 0.5 mCi 125I by the lactoperoxydase method. After two gel filtrations on Sephadex G-25 and on Sephadex G-75 in phosphate buffer with EDTA, Trasylol and mercapto-ethanol, a pure tracer with a specific activity of 140 microCi/micrograms was obtained. It remained utilizable for 30 days when kept at + 4 degrees C. Gel filtration on Aca 202 did not give good purification, while gel filtration on Aca 54 was good but slower than on Sephadex G-75. The binding to antibody in absence of unlabelled beta-LPH was 32% for an anti-beta-LPH antiserum diluted at 1/4000. The non specific binding was 2.5%.