家蚕抗菌肽-死亡素杂合肽基因在大肠杆菌中的克隆与表达

近年来的研究发现 ,抗菌蛋白在生物体非专一性防御系统有着重要的作用 ,已有数十种具有抗菌活性的多肽被分离 ,这些多肽可大致分为 3类 ,即含分子内二硫桥的抗菌肽 ;具有双亲α 螺旋结构的抗菌肽 ;以及富含某种氨基酸残基的抗菌肽[1 ] ,一般来说 ,这些抗菌肽具有分子量小 ,稳定性好 ,无细胞毒性 ,抗菌谱广等特点。多种抗菌肽的一级结构和二级结构已经确定[2 ] ,但作用机理仍不明了。一般认为可能存在两种作用模式 ,即 1)通过肽 脂膜相关作用杀菌 ;2 )通过受体介导的识别过程起作用[1 ] 。ＣｅｃｒｏｐｉｎＢ是一种较早从家蚕中分离得到 ,由 …     

家蚕抗菌肽-死亡素杂合肽基因在大肠杆菌中的克隆与表达

近年来的研究发现,抗菌蛋白在生物体非专一性防御系统有着重要的作用,已有数十种具有抗菌活性的多肽被分离,这些多肽可大致分为3类,即含分子内二硫桥的抗菌肽;具有双亲a-螺旋结构的抗菌肽;以及富含某种氨基酸残基的抗菌肽[1],一般来说,这些抗菌肽具有分子量小,稳定性好,无细胞毒性,抗菌谱广等特点.多种抗菌肽的一级结构和二级结构已经确定[2],但作用机理仍不明了.一般认为可能存在两种作用模式,即1)通过肽-脂膜相关作用杀菌;2)通过受体介导的识别过程起作用[1].     

Synthesis and Antibacterial Activity of Analogues of the N-Terminal Fragment of the Sarcotoxin IA Antimicrobial Peptide

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1–18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.     

Structure and Orientation of the Mammalian Antibacterial Peptide Cecropin P1 within Phospholipid Membranes

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly α-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospho lipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to acispositive transmembrane voltage difference. Taken together, the results further support a “carpet-like” mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.     

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。     

抗菌肽天蚕素B基因在纳豆芽胞杆菌中的表达

抗菌肽是生物体内经诱导产生的一类具有生物活性的小分子多肽。天蚕素B(Cecropin B)是最早从天蚕体内分离得到的一种热稳定的可溶性多肽,在已分离的众多抗菌肽中抗性较强。纳豆芽胞杆菌具有优良的益生特性,本研究选择枯草芽胞杆菌的一种表达载体p HT43,将抗菌肽天蚕素B基因导入纳豆芽胞杆菌中,验证其在目的菌中是否能够表达和稳定传代以及进行抗菌活性分析。结果表明,天蚕素B基因在纳豆芽胞杆菌中表达,并能稳定传代,能够提高纳豆芽胞杆菌的抑菌活性,抗金黄色葡萄球菌的活性优于干酪乳杆菌和枯草芽胞杆菌。本研究为该重组菌作为饲料添加剂的应用提供了技术基础。本文首次报道天蚕素B在纳豆枯草芽胞杆菌中表达。     

重组magainin抗菌肽表达条件的优化及其对抑菌活性的影响

对magainin抗菌肽重组酵母菌的摇瓶发酵条件进行了优化。结果表明 :当重组酵母菌在BMGY培养基中增殖至OD₆₀₀ 为 5～ 6时转接BMMY培养基 ;BMMY培养基pH值为 6.0 ,表达后期pH值控制在 3.0 ;2 6℃培养 48h ;补加 0.5 %甲醇的同时补加 3%营养液 ;可提高magainin抗菌肽的表达量。优化后摇瓶发酵的表达量可提高到 195 μg/ml。     

人精液凝固蛋白SgI-52的表达与抗菌活性研究

以人精囊腺cDNA为模板,用嵌套式PCR技术扩增出编码成熟人精液凝固蛋白I(semenogelin I,SgI)第85–136位氨基酸(SgI-52)的核苷酸序列并将其插入载体pMAL-p2X中,成功构建的表达载体pMAL-p2X/SgI-52转化大肠杆菌DH5α后,重组融合蛋白诱导表达于大肠杆菌周质中。经第X因子剪切及超滤处理,得到表达的重组SgI-52。质谱分析结果证明重组SgI-52为目的肽。重组人SgI-52对大肠杆菌ATCC25922标准株以及耐氨苄标准株ML-35P的最低抑制浓度MIC分别为32.45以及46.30?μg/mL。我们的结果及相关研究提示在人精液液化过程中人精液凝固蛋白I的不同降解产物可能行使不同的生物学功能,值得进行深入研究。     

Immobilization of Escherichia coli Cells by Use of the Antimicrobial Peptide Cecropin P1

An immobilization scheme for bacterial cells is described, in which the antimicrobial peptide cecropin P1 was used to trap Escherichia coli K-12 and O157:H7 cells on microtiter plate well surfaces. Cecropin P1 was covalently attached to the well surfaces, and E. coli cells were allowed to bind to the peptide-coated surface. The immobilized cells were detected colorimetrically with an anti-E. coli antibody-horseradish peroxidase conjugate. Binding curves were obtained in which the signal intensities were dependent upon the cell concentration and upon the amount of peptide attached to the well surface. After normalization for the amount of peptide coupled to the surface and the relative binding affinity of the antibody for each strain, the binding data were compared, which indicated that there was a strong preference for E. coli O157:H7 over E. coli K-12. The cells could be immobilized reproducibly at pH values ranging from 5 to 10 and at ionic strengths up to 0.50 M.     

改造后的抗菌肽AD基因在毕赤酵母中表达效价研究

通过PCR方法使抗菌肽AD基因中带有XhoⅠ和XbaⅠ酶切位点序列,再克隆到表达载体pPICZαA中,重组表达载体转化GS115,经重组酵母菌表达的抗菌肽AD,其N端去除了其它氨基酸。对重组抗菌肽培养条件进行优化,在YEPD中含葡萄糖2%,pH为75,重组酵母菌培养30h后,05%甲醇诱导42h,杀菌效价达1843,是改造前的31倍。最后重组抗菌肽经CMcellulose23柱层析得到纯化。     

家蝇天然抗菌肽的分离纯化与活性分析

目的:纯化得到有抗菌活性的家蝇天然抗菌肽。方法:以革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)的混合菌液,在未灭活的情况下对家蝇幼虫实施带菌针刺以诱导抗菌肽的大量表达。采用固相萃取(SPE),得到三个对大肠杆菌和金黄色葡萄球菌均有强烈抑制作用的组分Sp1、Sp3和Sp8。用反相高效液相色谱(RP-HPLC)对活性最强的Sp1进行了纯化,液体生长抑制法证明其中的组分Ⅲ具有最强活性。毛细管电泳(CE)显示该组分由两种物质组成。结果和结论:家蝇血淋巴中存在大量有抗菌活性的物质。该研究利用SPE和RP-HPLC与CE联用的方法,得到了有较强抗菌活性和较宽抗菌谱的粗提物,并获得一个推测由两种阳离子型抗菌肽组成的成分,为后期研究打下了基础。     

复合抗菌肽PL在毕赤酵母中的分泌表达及其活性研究

为了获得抗菌活性较强的抗菌肽,将几种抗菌肽串联起来在毕赤酵母中表达,并比较其与单独抗菌肽的抑菌活性。以GenBank中的Protegrin-1(PG-1)、ScorpionDefensin(SD)、Metalnikowin-2A和SheepMyeloidAntibacterialPeptide(SMAP-29)(序列号分别为AAB27599,AAAB27538、P80409和P49928)成熟肽段作为模板序列,根据巴斯德毕赤氏酵母(P.pastoris)偏好密码子,设计并人工合成复合抗菌肽pl基因,同时用SOE法获得ScorpionDefensin的基因,分别克隆到pPICZαA载体中,转化P.pastoris受体菌X-33,在醇氧化酶(AOX)启动子调控下,复合抗菌肽PL及SD均获得表达。体外抑菌试验检测复合抗菌肽PL与单独的蝎子防御素SD的热稳定性、酸稳定性、最低抑菌浓度等,结果显示复合抗菌肽PL及SD具有很强的热酸稳定性,而针对不同的细菌,复合抗菌肽则表现出了强于单独的SD的活性,特别是对大肠杆菌。上述结果说明了该复合抗菌肽具有很好的开发前景。     

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.     

天蚕素A-爪蟾素2杂合肽P18对MDA-MB-435s乳腺癌细胞的作用研究

目的 研究天蚕素A-爪蟾素2杂合肽P18对体外培养的人类乳腺癌细胞株MDA-MB-435s的活性作用.方法 采用MTT法检测P18对体外培养的MDA-MB-435s细胞增殖的影响;使用Live/Dead Viability/Cytotoxicity试剂盒对细胞进行染色,并在荧光显微镜下观察细胞的存活状态;通过DiBAC_4(3)染色,以荧光分光光度计检测给肽时细胞膜的膜电位变化;在透射电子显微镜下观察P18作用24 h后细胞超微结构的变化.结果 MTT试验表明P18对MDA-MB-435s细胞的增殖具有浓度依赖性的抑制作用;荧光显微镜下可观察到P18可以使MDA-MB-435s细胞膜破损,细胞死亡;P18作用时,荧光分光光度计检测到MDA-MB-435s的细胞膜发生了去极化现象;透射电子显微镜下可见大量细胞崩解坏死.结论 杂合肽P18能够引起MDA-MB-435s细胞膜的通透性改变而导致细胞坏死.     

天蚕素A-蜂毒素杂合肽用pBV220载体的表达、纯化和活性测定

为提高抗菌肽的表达,在抗菌肽的N端融合了1段酸性小肽以中和表达产物对宿主的毒性;并将融合肽基因同向串连成多拷贝,在大肠杆菌中获得了较高的表达。用化学合成法分别合成了编码天蚕素A(1-8)-蜂毒素(1-10)杂合肽和酸性小肽的DNA片段,首先将其拼接成融合肽的完整基因,然后通过前后接头将融合肽基因连接成两侧具有EcoRI和SalI酶切位点的同向串连的多拷贝基因。将5份拷贝的基因克隆至pBV220表达载体,转化E.coliDH5α,温度诱导得到表达量为35%的融合蛋白。表达产物主要以包涵体形式存在,将包涵体溶解,经Ni²⁺-NTA琼脂糖亲和层析获得纯化的融合蛋白。融合蛋白再经CNBr切割和阳离子交换层析,得到纯化的抗菌肽,经蛋白质N端测序确认序列正确。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。     

Antibacterial,Cytotoxicity and Mechanism of the Antimicrobial Peptide KR-32 in Weaning Piglets

International Journal of Peptide Research and Therapeutics - Diarrhea is a serious issue among livestock, and pathogens and viral infections are the main causes of diarrhea, especially in weaned...     

小羽贯众提取物抗菌活性的初步研究

目的：比较小羽贯众根状茎和叶提取物体外抑菌活性。方法：牛津杯法和96孔板法,通过测定抑菌圈大小、最小抑菌浓度（MIC）和最小杀菌浓度（MBC）,比较小羽贯众根状茎和叶提取物分别对10种常见细菌的抑制作用,以及不同产地抑菌效果的比较。结果：小羽贯众的根状茎和叶的提取物对10种常见的细菌均表现出较强的抑菌活性,肺炎克雷伯氏菌对提取物最敏感。结论：小羽贯众提取物作为一种天然的抗菌物质,具有广谱抗菌效果,有较高的应用潜力和开发前景。     

小羽贯众提取物抗菌活性的初步研究

目的:比较小羽贯众根状茎和叶提取物体外抑菌活性。方法:牛津杯法和96孔板法,通过测定抑菌圈大小、最小抑菌浓度(MIC)和最小杀菌浓度(MBC),比较小羽贯众根状茎和叶提取物分别对10种常见细菌的抑制作用,以及不同产地抑菌效果的比较。结果:小羽贯众的根状茎和叶的提取物对10种常见的细菌均表现出较强的抑菌活性,肺炎克雷伯氏菌对提取物最敏感。结论:小羽贯众提取物作为一种天然的抗菌物质,具有广谱抗菌效果,有较高的应用潜力和开发前景。     

新疆家蚕抗菌肽在毕赤酵母中表达及活性研究

目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性.方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽(Cecropin-XJ)基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoR Ⅰ及Not Ⅰ双酶切,构建重组表达质粒pPIC9K-(Cecropin-XJ),酶切及测序正确后,电转化到毕赤酵母GS115,对分泌表达的重组蛋白进行活性检测.结果:PCR扩增获得192 bp Cecropin-XJ,成功构建pPIC9K-Cecropin-XJ,优化诱导条件证明在pH 6的BMMY培养液中,0.5%甲醇诱导约48h后,获得的表达产物活性较强,对多种革兰氏阴性菌和阳性菌具有抗菌活性,在100℃条件下,其活性可维持100min以上.结论:新疆家蚕抗菌肽在毕赤酵母中分泌表达,为大规模发酵生产奠定了基础.     

Synthesis and Antibacterial Activity Evaluation of Biphenyl and Dibenzofuran Derivatives as Potential Antimicrobial Agents against Antibiotic-Resistant Bacteria

The escalating prevalence of antibiotic-resistant bacteria has led to a serious global public health problem; therefore, there is an urgent need for the development of structurally innovative antibacterial agents. In our study, a series of biphenyl and dibenzofuran derivatives were designed and synthesized by Suzuki-coupling and demethylation reactions in moderate to excellent yields (51–94% yield). Eleven compounds exhibited potent antibacterial activities against the prevalent antibiotic-resistant Gram-positive and Gram-negative pathogens, among which compounds 4′-(trifluoromethyl)-[1,1′-biphenyl]-3,4,5-triol (6i) and 5-(9H-carbazol-2-yl) benzene-1,2,3-triol (6m) showed the most potent inhibitory activities against methicillin-resistant Staphylococcus aureus and multidrug-resistant Enterococcus faecalis with MIC (minimum inhibitory concentration) values as low as 3.13 and 6.25 μg/mL, respectively. Compounds 3′,5′-dimethyl-[1,1′-biphenyl]-3,4,4′,5-tetraol (6e), 4′-fluoro-[1,1′-biphenyl]-3,4,5-triol (6g), and 4′-(trifluoromethyl)-[1,1′-biphenyl]-3,4,5-triol (6i) showed comparable inhibitory activities with ciprofloxacin to Gram-negative bacterium carbapenems-resistant Acinetobacter baumannii. Study of the structure–activity relationship indicated that a strong electron-withdrawing group on the A ring and hydroxyl groups on the B ring of biphenyls were beneficial to their antibacterial activities, and for benzo-heterocycles, N-heterocycle exhibited optimal antibacterial activity. These results can provide novel structures of antibacterial drugs chemically different from currently known antibiotics and broaden prospects for the development of effective antibiotics against antibiotic-resistant bacteria.