棉铃虫幼虫抗菌肽的初步研究*

棉铃虫(Heliothisarmigera)五龄幼虫经大肠杆菌、金黄色葡萄球菌混合菌诱导后,产生抗菌肽,经100℃热处理15min后活性不变,通过琼脂糖孔穴扩散法测定,对革兰氏阴性菌、革兰氏阳性菌以及真菌都具有很强的抑菌活性。CM-Sepharose离子交换层析粗纯化产物经SDS-PAGE测定,其分子量约3kD。     

产抑菌物质乳杆菌的筛选及性质的研究

从健康成年人的口腔中筛选出一株产抑菌物质的菌株HO-69,经API系统鉴定为植物乳杆菌。排除酸性末端产物与过氧化氢的影响后,HO-69的发酵上清液对变形链球菌等革兰氏阳性菌、大肠杆菌等革兰氏阴性菌具有抑制作用。抑菌物质经初步提取,推测其分子量在10kDa以下,胰蛋白酶处理后抑菌活性部分损失,100℃水浴加热20min依然保持较高的抑菌活力,显示活性的pH范围是3.0~7.0,活力随pH的降低而增强。     

筛选产类细菌素乳酸菌及类细菌素特性研究

从健康鸡肠道内容物及新鲜粪便中分离到21株乳酸菌,通过单层琼脂平板扩散实验,筛选出1株对藤黄微球菌(Micrococcus luteus)、金黄色葡萄球菌(Staphylococcus aureus)和枯草杆菌(Bacillus subtilis)等革兰氏阳性菌和大肠杆菌(Escherichia coli)等革兰氏阴性菌有明显抑菌活性代谢产物的乳酸菌,经细菌鉴定为乳杆菌属。排除酸和过氧化氢的干扰后,发酵液上清对指示菌仍有抑菌活性;用胰蛋白酶和蛋白酶K处理后抑菌活性明显降低,而胃蛋白酶对其活性无影响;用过氧化氢酶作用上清液,抑菌效果不变,说明过氧化氢未起作用;pH在2.0~7.0时,发酵上清液有抑菌活性;培养液粗提物经120℃处理20 min仍有部分活性,表明培养上清中有蛋白质类细菌素。     

重组Hepcidin融合蛋白的氧化复性及活性鉴定

将诱导表达的His-hepcidin融合蛋白包涵体通过固定化金属离子配体亲和层析(IMAC)柱分离纯化后,在cys-teine/cystine氧化还原体系中氧化形成二硫键,稀释复性后用肠激酶将融合蛋白的his-tag切除。酶切后所得的Hepcidin经抑菌圈试验检验,对枯草芽孢杆菌等革兰氏阳性菌和部分革兰氏阴性菌具有抗菌活性。     

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。     

融合表达HLH结构域对猪溶菌酶抗菌活性的影响

[目的]通过遗传操作提高猪溶菌酶抗革兰氏阴性菌的活性。[方法]对猪溶菌酶进行分子模拟,得到了包含功能肽的螺旋-回环-螺旋(HLH)结构域,将HLH编码基因与猪溶菌酶基因N端或C端进行融合,于大肠杆菌中诱导表达,经复性、纯化后检测其抗菌活性,并利用原子力显微镜和荧光染色对抗菌活性较高的融合蛋白杀菌机制进行初探。[结果]与对照组相比,N端和C端融合蛋白基本保持了猪溶菌酶对革兰氏阳性菌的活性;同时,两种融合蛋白对革兰氏阴性菌的抗菌活性均显著增强,其中N端融合产物活性更高,它对大肠杆菌ATCC 10798、大肠杆菌ATCC 25922、克雷伯氏菌TR5、铜绿假单胞菌ATCC 15442、沙门氏菌CMCC(B)50335的抗菌系数分别为1.64、1.24、2.56、1.72和1.42,最低抑菌浓度分别为90μg/mL、100μg/mL、40μg/mL、80μg/mL和100μg/mL。经检测,该融合蛋白能显著增强革兰氏阴性菌细胞膜的通透性。[结论]通过融合表达自身来源的HLH结构域,猪溶菌酶抗革兰氏阴性菌活性得到了显著提升,可为其它溶菌酶抗菌活性的提高提供参考。     

动物乳杆菌的分离鉴定及其抑菌蛋白的特性分析

通过滤纸片法从健康肉猪猪大肠、小肠中分离得到212株抗生物质产生菌, 以杯碟法复筛, 得到1株对溶壁微球菌(Micrococcus lysodeikticus)、金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、蜡状芽孢杆菌(Bacillus cereus)等革兰氏阳性菌和大肠杆菌(Escherichia coli)、鼠伤寒沙门氏菌(Salmonella typhimurium)等革兰氏阴性菌以及部分真菌如禾谷镰刀霉(Fusarium graminearum)均有强烈抑制作用的乳酸菌。经形态学、生理生化特征及16S rDNA序列同源性分析等手段鉴定该菌株为动物乳杆菌(Lactobacillus animalis)。排除酸和过氧化氢的干扰后, 该菌株的发酵上清液对指示菌仍有明显抑菌活性; 用蛋白酶处理该菌株的发酵上清液后, 抑菌活性丧失; 发酵液粗提物具有较好的热稳定性(经121°C处理20 min仍有较强抑菌活性)以及较宽的抑菌活性pH值范围(3.5~5.5), 因此初步认为该菌株产生一类具有广谱抑菌活性的类细菌素物质。     

一种猪溶菌酶来源的抗菌六肽的分离鉴定及其性质

为提高猪溶菌酶(Sus scrofa lysozyme,SSL)的抗革兰氏阴性菌活性,将其进行了不同蛋白酶的水解,选择抗革兰氏阴性菌效果最好的水解产物,利用凝胶过滤色谱和反相制备色谱进行分离,对其功能成分进行液质联用鉴定。对分离得到的物质进行抗菌活性验证和生物信息学的分析,并在此基础上对抗菌物质的杀菌机理进行了探讨。结果表明,胰蛋白酶的水解产物具有较高的杀灭革兰氏阴性菌的活性,进一步分离纯化得到了具有抗革兰氏阴性菌活性的六肽A-W-V-A-W-K。经化学合成验证,该六肽既保留了SSL的部分抗菌活性,也具备杀灭多种革兰氏阴性菌的能力。进一步分析发现其位于SSL分子C端的一个螺旋-回环-螺旋的结构中,并由此推测其杀菌机理是通过改变细胞膜的渗透性,进而使细胞内溶物流出而造成细胞死亡,而抗菌实验也验证了这一推测。该抗菌肽的发现为后续提高SSL的抗菌活性提供了理论依据。     

鸡源抗菌肽Fowlicidin-2在大肠杆菌中的 重组表达及其生物学活性鉴定

【目的】对抗菌肽Fowlicidin-2基因进行克隆与表达,并鉴定其生物学活性。【方法】根据抗菌肽Fowlicidin-2氨基酸序列,依照大肠杆菌(E.coli)密码子的偏爱性,人工设计合成其编码基因。与质粒pET-32a连接,构建重组表达载体,转化表达宿主菌E.coliBL21(DE3),IPTG诱导表达,融合蛋白经溴化氰裂解后进行纯化,测定重组抗菌肽的抑菌活性。【结果】Fowlicidin-2融合蛋白以包涵体形式表达,经溴化氰裂解后,成功释放出Fowlicidin-2,获得的重组Fowlicidin-2对革兰氏阳性菌和革兰氏阴性菌均有明显的抑菌效果。【结论】实现了抗菌肽Fowlicidin-2的重组表达,为抗菌肽的重组量化制备提供了理论基础与技术手段。     

黄粉虫幼虫小分子量抗茵肽的分离纯化

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TenebriomolitorL．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、SuperdexPeptide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Resource15RPC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1876．21u、1904．21u的小肽组成,可能是一种比Thanatin分子量更低的昆虫抗菌肽。     

一种新融合抗菌肽Hex-Mag基因的克隆、表达及其抗菌活性的研究

通过在Magainin1-12(GIGKFLHSAKKF)的N端添加碱性氨基酸片段Hexapeptide(RRWQWR)以增强其对细胞膜的吸附能力来提高Magainin1-12的抗菌活性。利用Hexapeptide和Magainin1-12的基因序列,结合酵母偏爱密码子设计出新的融合基因Hex-Mag,通过重叠区扩增基因拼接法(Gene splicing by overlap extension,gene SOEing)利用PCR扩增出基因片段,再将融合基因进行酶切并纯化后导入穿梭质粒pPIC9中,构建受乙醇氧化酶1基因(AOX1)的启动子与转录终止区控制的酵母表达质粒,转化GS115毕赤酵母宿主菌,经表型筛选,阳性克隆用甲醇诱导表达。表达出的融合肽Hex-Mag分子量约2.3kDa,其耐热性强,在100℃条件下,其活性可维持3h以上。琼脂糖孔穴扩散法检测显示Hex-Mag对多种革兰氏阴性菌和阳性菌具有抑制活性,与Magainin1-12相比,其活性有明显增强,N端正电荷增加的预期效应得到初步体现。     

家蝇幼虫抗菌肽MDL-2对细菌细胞渗透性及代谢功能影响

研究了家蝇幼虫抗菌肽MDL-2与细菌相互作用时,抗菌肤MDL-2对细菌细胞壁的溶解作用、细胞膜渗透性和代谢的影响.抗菌肽MDL-2在抗菌过程中首先与细菌的细胞壁相互作用,使其破裂,抗菌肽对革兰氏阴性细菌大肠杆菌细胞壁的作用有浓度依赖性,而对革兰氏阳性细菌金黄色葡萄球菌MDL-2在较低的浓度时即可发生细胞壁破坏作用;抗菌...     

Identification and isolation of a bactericidal domain in chicken egg white lysozyme

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram-positive and Gram-negative bacteria. Fractionation of clostripain-digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I-V-S-D-G-N-G-M-N-A-W-V-A-W-R (amino acids 98-112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I-V-S-D-G-N-G-M-N-A-W-V-A-W-R. The peptide I-V-S-D-G-N-G-M consisting of the eight amino acids of the N-terminal side had no bactericidal properties, whereas the peptide N-A-W-V-A-W-R of the C-terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R-A-W-V-A-W-R) increased the bactericidal activity considerably. The D enantiomer of R-A-W-V-A-W-R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.     

Antimicrobial activity of a bovine hemoglobin fragment in the tick Boophilus microplus.

Antifungal and antibacterial activities were detected in the hemolymph and gut contents of the cattle tick, Boophilus microplus. A peptide with antibacterial activity from the tick gut contents was purified to homogeneity by reversed-phase chromatography. The molecular mass of the purified peptide was 3,205.7 Da, measured by matrix-assisted laser desorption/ionization mass spectrometry. The amino acid sequence was obtained by Edman degradation and showed that the peptide was identical to a fragment of the bovine alpha-hemoglobin. A synthetic peptide based on the sequence obtained showed characterization data identical to those of the isolated material, confirming its structure. The synthetic peptide was active in micromolar concentrations against Gram-positive bacteria and fungi. These data led us to conclude that the antibacterial activity detected in tick gut contents is the result of enzymatic processing of a host protein, hemoglobin. This activity may be used by ticks as a defense against microorganisms.     

家蝇幼虫抗菌肽MDL-2的电泳制备及其抗菌作用机理

通过体壁损伤和感染大肠杆菌同时诱导家蝇Musca domestica幼虫产生免疫血淋巴,经沸水浴热变性,透析浓缩处理,然后经Tricine-SDS-PAGE得到诱导前后家蝇幼虫血淋巴中蛋白差异表达条带,将该条带电泳回收,复性,抗菌活性检测等步骤,分离纯化得到抗菌肽MDL-2,其分子中富含Pro,Gly和碱性氨基酸,分子量为11 kD,对革兰氏阴性菌Escherichia coli和革兰氏阳性菌Staphylococcus aureus均有较强抗性,因此电泳制备抗菌肽的方法为此类生物微量活性物质的分离纯化提供一种行之有效的途径。通过MDL-2对大肠杆菌和金黄色葡萄球菌通透性和透射电镜超微结构的图谱分析,MDL-2首先与细菌的外膜结合,然后抗菌肽形成柔性的两亲空间构象与细胞内膜作用,扰乱了膜脂分子的排列,改变了细胞膜的通透性,影响细胞膜的结构和功能,细胞膜上形成了许多孔道,同时造成细胞内的原生质扩散,并从孔道向胞外渗漏,影响了细菌的代谢系统,最终引起细胞膜破碎,细胞完全解体,从而起到抑菌杀菌作用。     

Antibacterial acitivity of the peptide complex isolated from the preparation of leukocytic interferon

A complex of low-molecular cationic peptides having an anti-bacterial effect with respect to Gram-positive and Gram-negative bacteria was isolated from the preparation of leukocytic interferon. The antibacterial action of the peptide complex was experimentally studied in vitro. The study revealed that the degree of the antibacterial activity of the peptide complex depended on the concentration of the bacterial culture under study, the ionic power of the incubation medium and did not depend on the presence of the products of bacterial vital activity in the growth medium. The antibacterial action of the peptide complex on the test cultures of Gram-positive and Gram-negative bacteria, as well as on the cultures of bacteria isolated from patients with infectious inflammatory diseases of the organs of the urinary system, was established. These results opened prospects for the development of fundamentally new antibacterial preparation on the basis of the peptide complex obtained in our studies.     

粉纹夜蛾离体细胞抗菌肽的抗菌谱测定

用热灭活的大肠杆菌DHSQ诱导粉纹夜蛾(Trichoplusia ni)离体细胞产生抗菌肽,用三氯乙酸沉淀法提取出该活性物质,采用琼脂糖孔穴扩散法和生长抑制测定法测定其抗菌谱,发现该抗菌物质具有较广的抗微生物活性,其中特别是对革兰氏阴性菌中的沙门氏茵和大肠杆茵,酵母菌中的白色念珠菌,植物病源真茵中的花生白绢病茵和小麦赤霉病茵具有较强的抑菌活性,从而表明该物质是一种既抗细菌,又抗真菌的抗微生物肽。     

家蚕抗菌肽CM4对大肠杆菌超微结构影响的研究*(简报)

The effect of antibacterial peptide CM4 of Bombyx mori against E. coll K12 was investigated using scanning electron microscopy(SEM) and transmission electron microscopy (TEM). The ultrastructural changes of E. coli K12 were observed by the challenge of the purified antibacterial peptide CM4. The results showed that the antibacterial peptide caused a series of pathological changes on E. coli. SEM and TEM revealed aggregates of bacteria and SEM revealed wrin-kled bacterial surfaces in the early stage. Thereafter, plasmolysis was observed with irregular holes appearing in the two ends of bacteria and the cytoplasmic contents of the cells leaking out. Finally, bacteria became empty vesicles and disintegrated into small fragments subsequently. Comparatively, the bacterial membrane was normal and the bacterial structure remained intact in the control group.     

家蚕抗菌肽CM4对大肠杆菌超微结构影响的研究（简报）

The effect of antibacterial peptide CM4 of Bombyx mori against E. coli K12 was investigated using scanning electron microscopy(SEM) and transmission electron microscopy (TEM). The ultrastructural changes of E. coli K12 were observed by the challenge of the purified antibacterial peptide CM4. The results showed that the antibacterial peptide caused a series of pathological changes on E. coli. SEM and TEM revealed aggregates of bacteria and SEM revealed wrinkled bacterial surfaces in the early stage. Thereafter, plasmolysis was observed with irregular holes appearing in the two ends of bacteria and the cytoplasmic contents of the cells leaking out. Finally, bacteria became empty vesicles and disintegrated into small fragments subsequently. Comparatively, the bacterial membrane was normal and the bacterial structure remained intact in the control group.