乳酸菌素基因PlnE和PlnF在大肠杆菌中的异源表达

为获得高产量纯品植物乳杆菌素PlnE和PlnF,研究其开发生物兽药的可能,试验通过人工合成植物乳杆菌素基因PlnE和PlnF,构建表达载体pGEX-4 T-E和pGEX-4T-F,经IPTG诱导表达条件优化后,进行亲和层析纯化融合表达蛋白,并使用SDs-PAGE进行鉴定.试验结果表明,pGEX-4T-E和pGEX-4T-F构建成功,纯化后获得2种产量高达30%的纯品融合蛋白,SDS-PAGE鉴定两融合蛋白表达正确.说明PlnE和PlnF基因片段编码的多肽能在原核细胞中正确表达,为进一步研究该细菌素的生物活性奠定了基础.     

乳酸菌素基因PlnE和PlnF在大肠杆菌中的异源表达

为获得高产量纯品植物乳杆菌素PlnE和PlnF,研究其开发生物兽药的可能,试验通过人工合成植物乳杆菌素基因PlnE和PlnF,构建表达载体pGEX-4T-E和pGEX-4T-F,经IPTG诱导表达条件优化后,进行亲和层析纯化融合表达蛋白,并使用SDS-PAGE进行鉴定。试验结果表明,pGEX-4T-E和pGEX-4T-F构建成功,纯化后获得2种产量高达30%的纯品融合蛋白,SDS-PAGE鉴定两融合蛋白表达正确。说明PlnE和PlnF基因片段编码的多肽能在原核细胞中正确表达,为进一步研究该细菌素的生物活性奠定了基础。     

雏鸡β-抵御素具有抗食源病原活性

在发展中国家，食源性致病菌与大多数的食品污染有关。同时，此类细菌还会污染包括雏鸡在内的多种家禽制品。目前，人们对雏鸡消化道内的β-抵御素及其作用功效还知之甚少。荷兰乌特勒支大学兽医学院传染病学和免疫学系针对雏鸡β-抵御素gallinacin-6（Gal-6）的表达及其抗食源病原活性进行了研究。他们采用RT—PCR的方法，在食道中检测到大量表达的Gal-6mRNA，     

枯草芽孢杆菌细菌素的分离、表达及稳定性分析

旨在分离枯草芽孢杆菌及其所产细菌素,并分析重组表达后的细菌素的抑菌活性和稳定性。通过牛津杯扩散法筛选羊驼粪便中高产细菌素的芽孢杆菌;借助16S rRNA鉴定分离菌;采用硫酸铵沉淀、氯仿抽提、SDS-PAGE、质谱分析等技术获得细菌素的氨基酸序列;利用大肠杆菌表达系统对细菌素进行体外表达,并利用牛津杯扩散法测定其抑菌活性和稳定性。结果显示,分离菌是一种枯草芽孢杆菌,命名为枯草芽孢杆菌SXAU18,其可产生抑制金黄色葡萄球菌、表皮葡萄球菌、藤黄微球菌和单增李斯特菌生长的细菌素;枯草芽孢杆菌SXAU18所产细菌素可能是分子量为10～20 ku的DarA蛋白和两种未知蛋白,属于类细菌素的范畴,将未知蛋白分别命名为BLIS SXAU181和182;经原核表达的BLIS SXAU181和182主要以可溶性上清蛋白形式表达,纯化后为单一条带;重组BLIS SXAU181蛋白没有抑菌活性,重组BLIS SXAU182蛋白具有良好的抑菌活性,且具有耐高温、耐酸碱、耐人工胃液和肠液的的特性。综上,本研究从枯草芽孢杆菌SXAU18分离到具有抑制革兰阳性菌生长活性的细菌素,且重组表达后的BLIS SXAU18...     

发酵基质对枯草菌素Subticin 112产生规律的影响及发酵参数优化

寻求枯草菌素Subticin 112产生的最佳发酵参数,阐明不同发酵基质对细菌素产生规律的影响,同时最大限度减少有机氮源,为细菌素的分离纯化减少工序和成本,从而为Subticin 112的高效生产提供理论依据。利用响应面法中的部分因析设计(FFD)与中心组合试验(CCD)相结合,确定发酵基质中各组分最适宜配比;比较不同发酵基质对菌体生物量、细菌素效价、比生产速率、比生长速率的影响。结果表明:发酵基质中有机氮源含量降低了74.3%,总氮源含量降低了56%,细菌素活性提高了170%,相当于青霉素当量163.8 mg/L。细菌素最大比生产速率提高了184%。证实了微量元素锰与Subticin 112的产量有密切关系,Subticin 112以菌体的混合代谢模式产生。     

信号肽对溶血素在大肠杆菌中跨膜转运的影响

信号肽在引导外源蛋白的跨膜转运过程中具有重要作用。本试验通过PCR方法获得带有信号肽序列和不带有信号肽序列的溶血素基因,构建表达载体在大肠杆菌中进行诱导表达。经过溶血试验和免疫印迹试验证明缺失了信号肽的重组溶血素不能被跨膜转运至细胞外;带有信号肽的重组溶血素能够跨膜转运大肠杆菌细胞外。本试验为猪链球菌2型基因缺失弱毒疫苗的研究、细菌信号肽以及基因工程重组蛋白的可溶性表达进行了有益探索。     

mgrB和mcr-1在耐多黏菌素猪大肠杆菌中的作用

大肠杆菌mgrB变异和携带mcr基因是其对多黏菌素耐药的重要机制,威胁着多黏菌素的临床应用.为了比较mgrB和mcr在猪源大肠杆菌中对多黏菌素耐药的影响,本试验收集165株猪源大肠杆菌,采用微量肉汤稀释法测定菌株对多黏菌素敏感性,并对耐药菌的mcr-1～mcr-5以及mgrB进行检测及测序比对分析.结果显示,165株猪...     

细菌素及其应用的研究进展

细菌素是细菌自身生产的一类杀菌物质,由基因编码、核糖体合成,是自然界中细菌用于调节自身菌群结构的一类重要活性物质。细菌素的来源广泛、结构多样,对各种动物致病菌都具有高效抑杀作用,这些特点为细菌素开发成为一种新型抗菌药物提供了可能。作者对近年来在细菌素生物学特征的发展、分类更新及其应用研究等进行了综述,希望能为中国新型细菌素类动物药物的开发提供参考。     

美人鱼发光杆菌溶血素Hly_(ch)基因的原核表达及免疫血清制备

为研究美人鱼发光杆菌溶血素的携带情况及溶血素的免疫原性,对前期分离的美人鱼发光杆菌的溶血素基因进行了分析,同时进行了原核表达和免疫原性分析。结果显示,该菌携带溶血素Hlych基因,生物信息学分析,该基因与美人鱼发光杆菌溶血素基因同源性达到99%以上,细胞定位预测为胞外蛋白,且具有良好的免疫原性。研究结果提示该菌携带Hlych基因,细菌毒力增强,同时Hlych蛋白有一定的免疫原性,有望用于建立ELISA诊断方法。     

细菌素Bacteriocin E50-52(H)基因设计 及其毕赤酵母表达载体构建

设计及合成细菌素Bacteriocin E50-52(H)基因,克隆到组成型分泌表达质粒pGAPZαA中构建重组质粒pGAPZα-Bacteriocin E(H),经PCR、测序验证正确后,电转化整合到毕赤酵母基因组,基因组PCR、测序验证结果表明成功构建了细菌素组成型重组表达载体,为在毕赤酵母中表达奠定了基础。     

Diphenylamine increases cloacin DF13 sensitivity in avian septicemic strains of Escherichia coli.

Thirteen avian septicemic isolates of Escherichia coli were examined for the presence of the aerobactin iron transport system. All of the strains possessed a functional aerobactin system and hybridization experiments showed that the aerobactin genes were located on ColV-type plasmids in all cases. The expression of the aerobactin receptor IutA was also studied by determining the bacterial susceptibility to the bacteriocin cloacin DF13. Twelve of the 13 isolates were cloacin-resistant but became sensitive to this bacteriocin upon treatment with diphenylamine which caused a reduction in the amount of O-side chain lipopolysaccharide.     

Heterologous expression of gassericin A,a bacteriocin produced by Lactobacillus gasseri LA39

Gassericin A, a bacteriocin produced by Lactobacillus gasseri LA39, has a cyclic structure linking N‐ and C‐terminal amino acids. Gassericin A was expressed in Escherichia coli JM109 as a biotinylated fusion protein on the basis of the DNA sequence of mature bacteriocin. A positive clone accumulated the bacteriocin, with no activity, as a soluble fusion protein in the cytoplasm. After release of an N‐terminal tag with factor Xa protease, gassericin A was converted into an active peptide having N‐ and C‐termini. The total amount of purified bacteriocins (expressed and native) was 480 µg/L and 370 µg/L, respectively. However, the specific activity of expressed gassericin A was 15 AU/mg lower than that of native bacteriocin (2600 AU/mg). Although the actual Mr (molecular weight) of the expressed bacteriocin should be 5666, the peptide showed the same mobility (Mr 3800) in sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) as native cyclic gassericin A, suggesting that the expressed peptide retains compact folding of the molecule similar to that of native gassericin A.     

喷雾干燥细菌素的生物学活性研究

为了初步研究前期试验筛选获得的枯草芽孢杆菌产细菌素的生物学活性,本试验采用杯碟扩散法绘制出硫酸黏杆菌素和杆菌肽锌两种抗生素的抑菌效价标准曲线,用抗生素效价当量来评价细菌素的活性,并在不同温度处理下测定其抗菌活性和热稳定性。结果表明,125 mg/mL细菌素溶液抑菌效价与26 mg/mL硫酸黏杆菌素或11.9 mg/mL杆菌肽锌溶液相当。此外,该细菌素在高温条件下(60~80 ℃)处理15 min仍能维持较高抑菌活性。综上所述,本试验在细菌素和抗生素的抑菌活力之间建立了更直观的联系,并证明了该细菌素具有较好的热稳定特性,为其产业化推广提供了理论支持和技术借鉴。     

Effect of yeast with bacteriocin from rumen bacteria on laying performance,blood biochemistry,faecal microbiota and egg quality of laying hens

The purpose of this study was to evaluate the effect of yeast with bacteriocin from Ruminococcus albus 7 (albusin B) on physiological state and production performance of laying hens. One hundred and twenty 26‐week‐old Single Comb White Leghorn (Hyline) laying hens were assigned into five groups including: (i) control group, (ii) yeast control (YC), (iii) 0.125% yeast with bacteriocin (0.125B), (iv) 0.25% yeast with bacteriocin (0.25B) and (v) 0.5% yeast with bacteriocin (0.5B). All supplements were added to the experimental diets of the hens from 26 to 46 weeks of age. Samples were collected every 4 weeks. Blood samples were collected from the wing vein for blood biochemical parameters assay, and faecal samples were collected by swab for the microbiota test. The egg production performance was recorded daily, and fresh eggs were collected for quality test. The blood biochemical assay results indicated that the addition of yeast with bacteriocin decreased the AST (aspartate aminotransferase) activity and it also affects the lactate concentration in laying hen blood. The result of egg quality indicated that yeast with bacteriocin supplementation had no effect on the mass of yolk and the strength of eggshell, but it had positive effect on the laying performance under hot environment. Low concentration bacteriocin (0.125B) supplementation could decrease total yolk cholesterol. The faecal microbiota result indicated that the supplementation of bacteriocin increased the lactobacilli counts. The yeast with bacteriocin supplementation significantly decreased the clostridia counts under hot environment condition, especially in hens receiving 0.25B. Combining the data from clinic chemistry, faecal microbiota, egg production and egg quality, the 0.25B supplementation may result in the best physiological parameter and egg production performance of laying hen.     

Study on the Biological Activity of a Spray Dried Bacteriocin

The standard curve of two antibiotics (colistin sulfate and bacitracin zinc) was carried out to assess antimicrobial activity of bacteriocin,which obtained from our earlier work,using cup-dish method.In addition,we also observed the effects of the reaction temperature on the antibacterial activity of the bacteriocin.Results showed that 125 mg/mL bacteriocin displayed similar antibacterial activity to 26 mg/mL colistin sulfate or 11.9 mg/mL bacitracin zinc.Furthermore,the bacteriocin remained good thermal stability under high temperature (nearly 60 to 80 ℃) in 15 minutes.Overall,this research built a direct relationship between this bacteriocin and antibiotics,and testified good stablility of this bacteriocin,providing theoretical support and technology reference to promote its large scale production.     

Production of bacteriocin by Leuconostoc mesenteroides 406 isolated from Mongolian fermented mare's milk,airag

The purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides strain 406 that was isolated from traditional Mongolian fermented mare's milk, airag, were carried out. Leuconostoc mesenteroides strain 406 was identified on the basis of its morphological and biochemical characteristics and carbohydrate fermentation profile and by API 50 CH kit and 16S ribosomal DNA analyses. The neutral‐pH cell‐free supernatant of this bacterium inhibited the growth of several lactic acid bacteria and food spoilage and pathogenic organisms, including Listeria monocytogenes and Clostridium botulinum. The bacteriocin was heat‐stable and not sensitive to acid and alkaline conditions, but was sensitive to several proteolytic enzymes such as pepsin, pronase E, proteinase K, trypsin, and α‐chymotrypsin, but not catalase. Optimum bacteriocin production (4000 activity units/mL) was achieved when the strain was cultured at 25°C for 24–36 h in Man Rogosa Sharpe medium. The bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut‐off MW: 1000), and gel filtration chromatography. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that the bacteriocin had a molecular weight of approximately 3.3 kDa. To our knowledge, this is the first report of the isolation of a bacteriocin‐producing Leuconostoc strain from airag. An application to fermented milks would be desired.     

产细菌素乳酸菌的分离与筛选

本试验从国内多种发酵材料中分离培养出5株乳酸菌(L1、L2、L3、L4、L5),该5株乳酸菌发酵上清液均对大肠杆菌(G-)、枯草芽孢杆菌(G+)有显著抑制作用。在排除有机酸、过氧化氢等因素对抑菌效果的影响后,初步确定5株乳酸菌发酵上清液中的抑菌物质为细菌素,且该细菌素对非蛋白酶类、温度和有机溶剂不敏感。     

乳酸菌产生的新型抗菌物质——苯乳酸的抑菌性质及作用机理研究

苯乳酸是微生物产生的一种新型天然抗菌物质,已成为乳酸菌抑菌能力的有效标志之一。与乳酸菌产生的细菌素(如乳链球菌素)相比,苯乳酸是小分子物质、抑菌谱宽、稳定性高。作为一种新型生物防腐剂,苯乳酸在乳品工业中具有广阔的应用前景。研究表明苯乳酸对多种食源性致病菌如致病性大肠杆菌、单增李斯特菌和金黄色葡萄球菌,腐败菌包括产毒素的丝状真菌如赭曲霉、疣孢青霉和桔青霉,有很强的抑菌作用。目前的研究表明苯乳酸的作用靶位之一是微生物的细胞壁,但阐明苯乳酸的作用机理需要采用相关技术进一步研究其他作用位点。本文对苯乳酸的抑菌活性、作用机理等研究进展进行综述,并展望了今后的研究方向。     

枯草芽胞杆菌DarA蛋白的鉴定及其原核表达

目前,我国全面禁止饲料中添加抗生素,寻找新型的抗生素替代物是当前研究的热点之一.本研究旨在分离枯草芽胞杆菌SXAU18所产具有抗菌活性的蛋白物质,并通过原核表达系统获得其具有抗菌活性的重组蛋白.试验选用硫酸铵沉淀、氯仿抽提、分子截留等蛋白提取技术,对枯草芽胞杆菌SXAU18产生的抗菌蛋白进行分离纯化;通过SDS-PAG...     

Study on Physicochemical Properties of Lactobacillus plantarum DSM20174 Bacteriocin from Koumiss

To study the physicochemical properties extracted from koumiss of Lactobacillus plantarum DSM20174 bacteriocin,we purified the Lactobacillus plantarum DSM20174 bacteriocins and got the bacteriocin crude extractions after ammonium sulfate precipitation and dialysis.After purification with Sephadex G-100 gel column chromatography,bacteriocin specific activity apparently increased,reaching 154.70 AU/mL,yield was 43.5%.We detected the effects of purified Lactobacillus plantarum DSM20174 bacteriocin on bacteriostatic action of wild bovine pathogenic E.coli O78 by Oxford cup method under 3 different treatments.The results showed that:①In five groups of temperature treatments,the bacteriostatic action decreased with the increase of temperature,bacteriostatic actions of all groups were significantly different (P<0.05).Even after treatment at 121 ℃ 30 min,antimicrobial diameter was 14.90 mm.② After Lactobacillus plantarum DSM20174 bacteriocin were treated with pH 2.0 to pH 12.0,the higher of pH,the smaller of antibacterial diameter.The inhibitory effect of groups were significantly different (P<0.05) except pH 9.0 and pH 10.0.③Lactobacillus plantarum DSM20174 bacteriocins were treated by trypsin,pepsin and papain,antibacterial diameter apparently changed,bacteriostatic action was significantly different (P<0.05) before and after treatment.The results suggested that Lactobacillus plantarum DSM20174 bacteriocins were better antibacterial substances,but not thermal stability substances.It had a relatively wide pH range,and the smaller of pH,the stronger of its antibacterial activity,the antibacterial activity had been enhanced at optimum growth pH.It was not protease stability substances.