猪血中白细胞抗菌肽的提取及体外抑菌研究

为了研究猪血液白细胞抗菌肽对大肠杆菌、鸡源沙门杆菌等的体外抑菌活性,试验采用乙酸萃取、Superdex 30 PG凝胶层析纯化,收集组分并检测抑菌活性。结果表明:猪血液中抗菌肽对大肠杆菌、鸡源沙门杆菌均有良好的抑菌活性。说明该方法可用于猪血白细胞抗菌肽的提取并可抑制上述致病菌。     

奶牛子宫内膜粘液抗菌肽的分离纯化

为研发奶牛抗菌肽生物制剂,防治奶牛子宫内膜炎,本研究采用酸性尿素聚丙烯酰胺凝胶电泳琼脂糖弥散法检测子宫内膜粘液酸溶性提取物抗菌活性;应用琼脂糖电泳及水平电泳洗脱分离相应的抗菌条带,HPLC进一步分离纯化,琼脂糖弥散法检测纯化分子抗菌活性,Tricine-SDS-PAGE电泳检测其分子量。结果表明,从奶牛子宫内膜酸溶性提取物中纯化出一分子量约为14kD左右的抗菌多肽,对大肠杆菌(E.coliBL21)和金黄色葡萄球菌26003具有抗菌活性。本研究可能分离纯化出一种新的子宫内膜分泌的抗菌多肽,其可能参与奶牛子宫的天然防御机制。     

大口黑鲈不同组织中抗菌肽粗提及抑菌活性测定

研究了大口黑鲈不同组织中抗菌肽的提取及其粗提物对不同菌种的抑菌作用。以新鲜的大口黑鲈为试验材料,用5％乙酸作为提取液,对大口黑鲈粘液、皮肤、肝脏、脾、卵、鳃组织进行提取,并对粗提物进行抑菌活性检测。结果表明大口黑鲈粘液、皮肤和肝脏组织提取物对嗜水气单胞菌有抑菌作用,皮肤和肝脏组织提取物对大肠杆菌有抑菌作用。肝脏组织抗菌肽提取物经SephadexG-25凝胶过滤层析柱分离后,其收集液对嗜水气单胞菌仍具有抑菌作用。大口黑鲈皮肤和肝脏组织提取物对嗜水气单胞菌、大肠杆菌均有抑菌作用。     

乌鳢体表粘液抗菌肽CSM14的分离纯化及部分生物学活性研究

为建立分离纯化乌鳢体表粘液抗菌肽的方法并研究其抗菌活性,本研究通过嗜水气单胞菌对乌鳢进行刺激,采用甲醇提取、SephadexG-50凝胶层析和反向高效液相色谱从其体表粘液中提取抗菌肽,经质谱分析测定分子量;并对抗菌肽的抑菌谱、最小抑菌浓度(MIC)、热稳定性、溶血活性进行了初步研究。结果表明:分离纯化的乌鳢体表粘液抗菌肽分子量约为3024.15ku,并具有广谱的抗菌活性,热稳定性好,没有溶血活性。     

家蝇抗菌肽的分离纯化及生物学活性

将诱导家蝇提取的家蝇抗菌肽提取物，经凝胶过滤层析和离子交换层析，得到具有抗菌活性的单一峰，质谱结果显示抗菌肽分离物含有4种小肽，相对分子质量678．3～1017．2。家蝇抗菌肽对革兰氏阴性菌（大肠杆菌、巴氏杆菌）的抗菌活性优于革兰氏阳性菌（金黄葡萄球菌、枯草芽孢杆菌），对耐药性巴氏杆菌有较强的抗菌活性，对大肠杆菌的MIC为4．2～8．4mg／L，对耐药性巴氏杆菌的MIC为3．8～7．8mg／L。扫描电镜观察发现，家蝇抗菌肽通过损伤细胞壁使细胞质外流而达到杀菌的目的。家蝇抗菌肽对家兔、鸡、猪、小鼠、番鸭、鹅和家鸭红细胞无溶血反应和凝集反应。‘     

蜜蜂幼虫抗菌活性的初步研究

为了研究中华蜜蜂幼虫粗提取液的抗菌活性,试验通过诱导2日龄蜜蜂幼虫,分离、纯化出中华蜜蜂幼虫中的抗菌肽,研究其性质和抑菌特性。结果表明:中华蜜蜂幼虫提取液中含有抑菌活性物质,对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄八叠球菌均有明显的抑制作用,而对酵母和霉菌的生长没有明显抑制作用;此外,中华蜜蜂幼虫粗提取液分别经过高温、高盐、酸碱处理后仍保持较高的抑菌活性。说明蜜蜂幼虫的抑菌活性物质含有抗菌肽。     

枯草芽孢杆菌J-4菌株胞外产抑菌物质性质的初探

为了研究枯草芽孢杆菌(Bacillus subtilis)J-4菌株胞外产抑菌物质的性质,试验采用琼脂打孔扩散法,以大肠杆菌(Escherichia coli)为指示菌,考察硫酸铵盐析、透析处理、水浴处理、缓冲液pH值、蛋白酶、变性剂、离子种类等对该抑菌物质活性的影响。结果表明:J-4菌株胞外产生的抑菌物质可被80%饱和度的硫酸铵盐析;该抑菌物质的分子质量在8.0 ku以上;100℃水浴60 min,该抑菌物质仍有较高抑菌活性;在pH值为2.0~8.0范围内均有较高活性;抑菌物质分子结构中含有可被胃蛋白酶或胰蛋白酶酶解的肽键;抑菌物质的活性可被十六烷基三甲基溴化铵(CTAB)和高浓度的CaCl_2增强。说明枯草芽孢杆菌J-4菌株胞外产生的抑菌物质为抗菌蛋白类物质,具有较高的抗菌活性,热稳定性好,耐受鸡消化道pH值为4.0~7.0环境和消化酶,在养鸡业极具应用潜力。     

光滑爪蟾抗菌肽的分离纯化和对癌细胞的生长抑制作用

通过凝胶过滤层析及高效液相色谱法,从皮肤分泌物中分离出具有抑菌活性的物质,即光滑爪蟾抗菌肽。以大肠癌细胞为体外实验模型,通过细胞形态学、MTS试验,研究光滑爪蟾抗菌肽对SW480细胞的杀伤和生长抑制作用。结果表明,经过分离纯化,得到较纯的活性物质,即光滑爪蟾抗菌肽。通过细胞形态学观察,光滑爪蟾抗菌肽作用组中,细胞的数量明显减少,细胞明显皱缩,成圆形,细胞间隙增大,死细胞增多。MTS试验表明,光滑爪蟾抗菌肽对SW480的生长抑制作用随着浓度的增加而加强。     

Spinigerin α抗菌肽的分离纯化及其活性研究

Spinigerinα抗菌肽是一种源于白蚁的线性抗菌肽,包括25个氨基酸,不含半胱氨酸,呈α-螺旋结构。本研究采用凝胶加热-层析法,对Spinigerinα抗菌肽样液通过水浴加热至100℃保持20³0 min,除去不耐热的部分蛋白,经葡聚糖凝胶SephadexG-25脱盐层析分离得到Spinigerinα抗菌肽提纯物。根据SDS-聚丙烯酰胺凝胶电泳图谱绘制分子质量标准曲线,计算Spinigerinα抗菌肽的分子质量。试验结果表明:SDS-PAGE电泳表现为单一条带,其分子质量为2.74 ku;Spinigerinα抗菌肽对大肠杆菌、沙门氏菌、金黄色葡萄球菌均有抑菌作用,最小抑菌浓度分别为0.5 mg/mL、0.3 mg/mL、0.2 mg/mL。     

奶牛子宫内膜组织抗菌肽分离纯化及其抗菌活性研究

目的：研发奶牛抗菌肽生物制剂,防治奶牛子宫内膜疾病。方法：采用酸性尿素聚丙烯酰胺凝胶电泳和琼脂糖弥散法检测子宫内膜组织酸溶性提取物抗菌活性;应用琼脂糖电泳及水平电泳洗脱分离出相应的抗菌条带,HPLC进一步分离纯化,琼脂糖弥散法检测纯化物的抗菌活性,Tricine-SDS-PAGE电泳检测其分子量。结果：粗提物中有两种主要的抗菌成分,其对金黄色葡萄球菌26003、大肠杆菌BL-21、牛致病性大肠杆菌4605、绿脓杆菌10104具有抗菌活性。上层条带的主要色谱峰出现在43、45 min,其中45 min收集的样品有较强的抗菌活性,其相对分子质量约为14 k Da。下层条带主要色谱峰出现在5、35、43、45、46、47、50 min,其中35、43、45 min收集的样品有抗菌活性,其相对分子质量分别约为14-16、6和12 k Da。各个组分对大肠杆菌BL-21和金黄色葡萄球菌26003均具有抑菌活性。结论：从奶牛子宫内膜组织分离出了5个抗菌成分,从相对分子质量、抗菌活性等方面初步认定为抗菌多肽。     

Expression and Identification of Porcine Antimicrobial Peptide PBD-1 in Pichia pastoris

In order to produce antimicrobial peptide PBD-1 with bioactivity in Pichia pastoris,according to published amino acid sequence of PBD-1 and the partiality codon of yeast,the PBD-1 gene was amplified by SOE-PCR and cloned into pPIC9K to construct a recombinant expression vector pPIC9K-PBD-1.The recombinant vector was linearized by SacⅠ,and then transformed into SMD1168 by electroporation.Positive yeast expression strain was obtained by PCR.The antimicrobial peptide PBD-1 (approximately 4.5 ku) was expressed by methanol induction.Antibacterial activity assay showed that the antimicrobial peptide PBD-1 had better antibacterial activity against E.coli,S.aureus and B.subtilis.     

猪源抗菌肽PBD-1在毕赤酵母中的表达及鉴定

本试验旨在实现猪β防御素1 (poricine-β-defensin 1,PBD-1)在毕赤酵母中的表达,获得有抗菌活性的抗菌肽PBD-1。根据PBD-1的氨基酸序列和酵母密码子偏好性,设计优化其核苷酸序列,利用SOE-PCR技术获得PBD-1基因序列,克隆到毕赤酵母表达载体pPIC9K中,构建重组质粒pPIC9K-PBD-1,经SacⅠ线性化后转入毕赤酵母SMD1168中。PCR筛选得到阳性酵母表达菌株,经甲醇诱导后得到分子质量约4.5 ku的抗菌肽PBD-1。抗菌特性研究结果表明,表达产物抗菌肽PBD-1对大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌均有较好的抑制效果。     

新型黏虫抗菌肽AAP-1对大肠杆菌的抗菌作用

为评价一种新型黏虫抗菌肽（armyworm antimicrobial peptide 1，AAP-1）的抗菌活性，本试验首先采用固相化学合成法合成AAP-1，并通过高效液相色谱纯化和质谱检验，用抑菌圈法测定AAP-1对大肠杆菌的抗菌效果，倍比稀释法检测最小抑菌浓度（minimum inhibitory concentration，MIC），细菌平板计数法测定时间抗菌曲线；最后，通过超微量分光光度计评价AAP-1对大肠杆菌核酸泄露的影响，核酸凝胶电泳评价AAP-1对大肠杆菌胞内DNA的影响，透射电子显微镜观察评价AAP-1对大肠杆菌的整体作用效果。结果表明，合成的AAP-1纯度高于98%，分子质量为4 262.17 u；AAP-1对大肠杆菌具有良好的抗菌活性，MIC为7.8 μg/mL；对大肠杆菌的抗菌作用具有浓度依赖性，且在60 min内抗菌效果逐渐增强；AAP-1能够导致大肠杆菌核酸泄露，致使胞内DNA总量减少，且呈现浓度依赖性；AAP-1也会导致大肠杆菌细胞膜破坏、胞内电子密度明显降低、胞质溶解等现象。综上，本研究化学合成了一种新型抗菌肽AAP-1，且纯度高达98%，其对大肠杆菌具有良好的抗菌效果。本研究可为深入研究AAP-1对大肠杆菌的抗菌机制提供前期数据，可为APP-1的临床应用奠定理论基础。     

Discovery,Isolation,Identification and Characterization of Novel Non-ribosomal Peptide Antibacterial Active Substances in Bacillus

The aim of this study was to search for novel non-ribosomal peptide antimicrobial substances based on the screening of bacterial secondary metabolites.The bacteria isolated from soil,sea water and common marine organisms in Yantai coastal area were isolated and purified.E.coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were selected as indicator bacteria,and E.coli B2 (bla_NDM-5+mcr-1) and methicillin-resistant Staphylococcus aureus (MRSA) T144 were used as indicator for secondary screening.The genome was extracted and the PCR products were sequenced to determine the active species.The secondary metabolites of bacteria were extracted by organic extraction,purified by gel chromatography and preparative liquid chromatography,and purity was detected by analytical liquid chromatography.The results of sequencing showed that the active strain belonged to Bacillus amyloliquefaciens sp.,and was named as Bacillus amyloliquefaciens 9-14 (active bacterium 9-14).The results of antibacterial test showed that the metabolites of active bacterium 9-14 had high inhibitory effect on Staphylococcus aureus ATCC 29213,MRSA T144,E.coli ATCC 25922 and E.coli B2.The metabolites of active bacterium 9-14 were cyclic lipopeptides composed of amino acid chains,which belonged to the derivatives of ibuprofen.The biological characteristics and antibacterial spectrum of the metabolites of active bacterium 9-14 were studied.The results showed that the metabolites of active bacterium 9-14 had good thermal stability and acid-base stability.And the antibacterial activity of the antibacterial substance treated with trypsin,pepsin,protease K and papain was not significantly weakened and had good stability.The antibacterial substance also had inhibitory effect on Staphylococcus aureus and E.coli,but had no activity against Pseudomonas aeruginosa,Klebsiella pneumoniae,Enterococcus faecalis and Bacillus cereus.In this study,a new antibacterial substance was obtained,which could be used as the precursor of antibacterial drugs,and could provide certain reference for food safety and disease control.     

Antibacterial Effect of Novel Armyworm Antimicrobial Peptide (AAP-1) Against Escherichia coli

In order to evaluate the bactericidal activity of a novel armyworm antimicrobial peptide (AAP-1),the bactericidal effect of AAP-1 on Escherichia coli (E.coli) was investigated.Firstly,AAP-1 was synthesized by solid-phase chemically synthesis,and purified by high-performance liquid chromatography and tested by mass spectrometry.Then,the bactericidal effect of AAP-1 against E.coli was determined by the bacteriostatic circle method.The minimum inhibitory concentration (MIC) was detected by the double dilution method.The time kill curve was measured by bacterial plate counting method.Finally,the effect of AAP-1 on the nucleic acid leakage of E.coli was evaluation by ultra-micro spectrophotometer.The effect of AAP-1 on the intracellular DNA of E.coli was tested by nucleic acid gel electrophoresis.The overall effect of AAP-1 on E.coli was observed through transmission electron microscope.The results showed that the purity of the synthesized AAP-1 was more than 98%,and its molecular weight was 4 262.17 u.AAP-1 showed good antibacterial activity against E.coli,with a MIC of 7.8 μg/mL.AAP-1 had a concentration-dependent bactericidal effect on E.coli,and within 60 min the bactericidal effect gradually increased.AAP-1 could cause the nucleic acid leakage of E.coli,and resulted in a reduction in the total amount of intracellular DNA of E.coli with concentration-dependent.AAP-1 could also cause cell membrane destruction,significantly reduce intracellular electron density,resulted in cytoplasmic dissolution of E.coli,and so on.In summary,this study suggested that a novel antimicrobial peptide AAP-1 could be chemically synthesized with a purity of up to 98% and showed its good bactericidal effect on E.coli.This study could provide preliminary data for the in-depth study of the bactericidal mechanism of AAP-1 against E.coli and laid a theoretical foundation for the clinical application of AAP-1.     

芽孢杆菌中新型非核糖体肽类抗菌活性物质的发掘、分离鉴定及特性研究

本研究旨在基于对细菌次级代谢产物的筛选,寻找新型非核糖体肽类抗菌物质。通过对烟台沿海地区的土壤、海水及近海常见海洋生物中的细菌进行培养,分离纯化得到细菌的单克隆,以大肠杆菌（E.coli）ATCC 25922和金黄色葡萄球菌ATCC 29213为指示菌进行初步筛选,以耐多黏菌素和碳青霉烯E.coli B2（bla_NDM-5+mcr-1）和耐甲氧西林金黄色葡萄球菌（MRSA） T144作为指示菌对有活性的细菌进行二次筛选。通过提取基因组进行PCR扩增产物测序比对,确定活性菌种属。采用有机萃取法对细菌的次级代谢产物进行萃取,通过凝胶层析和制备液相色谱进行纯化,利用分析型液相色谱进行纯度检测,进—步利用质谱对纯化后的抗菌活性物质进行结构鉴定。测序结果表明,活性菌属于解淀粉酶芽孢杆菌种,将其命名为解淀粉酶芽孢杆菌9-14（活性菌9-14）。抑菌试验结果表明,活性菌9-14的代谢产物对金黄色葡萄球菌ATCC 29213、MRSA T144、E.coli ATCC 25922和E.coli B2均具有高效抑制作用。活性菌9-14代谢产物是由氨基酸链组成的环状脂肽,属于伊枯草菌素的衍生物。对活性菌9-14代谢产物的生物学特性及其抑菌谱研究发现,活性菌9-14的代谢产物具有良好的热稳定性和酸碱稳定性,该抗菌物质经胰蛋白酶、胃蛋白酶、蛋白酶K和木瓜蛋白酶处理后抗菌活性没有明显减弱,具有较好的稳定性;该抗菌物质对所用金黄色葡萄球菌和大肠杆菌同样具有抑制作用,对绿脓杆菌、肺炎克雷伯杆菌、粪肠球菌和蜡样芽孢杆菌均不表现活性。本研究得到一种新型的抗菌物质,以该抗菌物质为抗菌药物的前体,可为食品安全和疾病控制提供一定的参考依据。     

某肉鸡场鸡源大肠杆菌耐药性跟踪检测与分析

2012~2014年从辽宁某肉鸡养殖场采集300份鸡泄殖腔拭子,分离获得152株鸡源大肠杆菌。采用微量稀释法对152株鸡源大肠杆菌进行了14种兽用和16种人用抗菌药的药物敏感性测定,并参照CLSI肠杆菌判定标准进行判定。结果显示,鸡源大肠杆菌对兽用阿莫西林、氨苄西林、恩诺沙星、复方新诺明、磺胺异噁唑、四环素和人用哌拉西林、替卡西林、环丙沙星均产生明显耐药性,耐药率较高。对所分离的鸡源大肠杆菌耐药性检测结果与EUCAST发布的野生型菌株MIC值分布进行比较分析,耐药结果基本一致,但通过MIC值分布比较,结果显示出分离菌株的大观霉素、氨苄西林、恩诺沙星、复方新诺明、头孢噻吩、头孢呋辛、环丙沙星和妥布霉素药物的MIC值发生了明显右移,表明该肉鸡场大肠杆菌对上述抗菌药的耐药强度呈现逐渐增强的趋势。在养殖过程中应采取科学饲养和严格的生物安全措施防控疾病,科学合理使用抗菌药物,控制肉鸡场细菌耐药性的产生和传播。     

Tracking Detection and Analysis of Drug Resistance of Chicken-derived Escherichia coli in a Broiler Farm

From 2012 to 2014,300 chicken cloacal swab samples were collected from a broiler farm in Liaoning province,152 strains of chicken-derived Escherichia coli were isolated and identified.The 152 strains of chicken-derived Escherichia coli were detected on 14 kinds of veterinary antimicrobial drugs and 16 kinds of antimicrobial drugs for human by using trace dilution method,and reference to CLSI enterobacter decision criteria to determine.The results showed that chicken-derived Escherichia coli had obvious resistance to veterinary antimicrobial drugs (amoxicillin,ampicillin,enrofloxacin,trimethoprim-sulfamethoxazole,sulfafurazole,tetracycline) and antimicrobial drugs for human (piperacillin,ticarcillin,ciprofloxacin),the resistance rates were high.The resistance test results of isolated chicken-derived Escherichia coli comparing with the MIC value distribution of EUCAST wild type strains were same,but through comparing MIC value distribution,the results showed the isolates of spectinomycin,ampicillin,enrofloxacin,trimethoprim-sulfamethoxazole,cefalotin,cefuroxime,ciprofloxacin and tobramycin drugs MIC value moved to the right obviously,indicated that the antibiotic resistant strength of Escherichia coli increased gradually.The process of breeding and strict biosecurity measures should be taken scientifically to control diseases,and the antibacterial agents should be applied reasonably to control broiler farm production and propagation of bacterial drug resistance.     

植物乳杆菌抑菌蛋白的分离鉴定及抑菌特性研究

【目的】筛选新型广谱有效的抑菌蛋白,为全面解析植物乳杆菌抑菌机制和开发新型抗菌制剂奠定基础。【方法】以植物乳杆菌GX20200417-1为研究对象,采用牛津杯法测定其抑菌活性,以低温离心和超滤法初步分离获得胞外蛋白并测定其抑菌活性,对胞外蛋白进行不同温度、pH及蛋白酶处理,研究抑菌蛋白理化特性,利用液相色谱串联质谱(LC-MS/MS)进一步分析鉴定植物乳杆菌代谢产物中的抑菌成分。【结果】植物乳杆菌GX20200417-1对沙门氏菌、金黄色葡萄球菌和大肠杆菌均具有良好的抑菌作用;发酵上清液在发酵24 h的抑菌能力最强;胞外蛋白的抑菌能力与发酵上清液相近;植物乳杆菌抑菌蛋白具有较好的耐热特性,与对照组相比,在20~80 ℃时抑菌活性均无显著变化(P＜0.05),在pH 6.0~8.0时抑菌活性最佳,对蛋白酶敏感;经LC-MS/MS鉴定分析,检测出5种可信度较高且与抑菌作用相关的蛋白质,分别是片球菌素pediocin PA-1、溶菌素(lysin)、聚酮合酶(polyketide synthase)、辅助蛋白(accessory protein)和LysM peptidoglycan-binding domain-containing protein,分子质量分别为5.348、7.348、8.348、6.348和19.662 ku;蛋白质功能分析结果表明,片球菌素pediocin PA-1与溶菌素主要通过破坏细菌细胞壁与细胞膜,从而达到抑菌效果。LysM peptidoglycan-binding domain-containing protein可识别含有N-乙酰氨基葡萄糖(GlcNAc)残基的肽聚糖,上调抗菌肽的表达;辅助蛋白主要参与细菌素的合成,聚酮合酶主要参与抗生素的合成,二者通过参与抑菌物质的合成间接发挥抑菌作用。【结论】本研究成功分离并鉴定植物乳杆菌GX20200417-1的5种抑菌蛋白;植物乳杆菌GX20200417-1可能通过其代谢产物中的多种抑菌蛋白协同发挥抗菌作用。