乳酸片球菌R-4细菌素PA-1原核表达及其理化特性

为实现原核表达产出细菌素并检测其理化特性，作者将乳酸片球菌 R-4细菌素pedA基因进行扩增回收，与pMD19-T载体连接后转入E.coli DH5α感受态细胞进行克隆。提取克隆后的pedA基因与表达载体pET-32a（+）连接，形成重组质粒pET-32a-pedA并转入E.coli BL21(DE3)感受态细胞，经异丙基硫代半乳糖苷诱导，乳酸片球菌R-4细菌素PA-1在大肠杆菌细胞进行表达。表达蛋白质经Ni-NTA柱纯化后，以金黄色葡萄球菌为指示菌检测其理化特性。结果表明，在E.coli BL21(DE3)细胞中成功表达相对分子质量为26 000的乳酸片球菌R-4细菌素PA-1并完成纯化。纯化后的乳酸片球菌R-4细菌素PA-1在40~121 ℃作用20 min、在pH 2~12、紫外线照射0~10 h、过氧化氢酶作用2 h后，其抑菌范围分别为14.7~15.6 mm、14.0~16.5 mm、15.1~15.8 mm和14.9 mm，而分别经胃蛋白酶和胰蛋白酶作用2 h均失去抑菌作用。这表明乳酸片球菌R-4细菌素PA-1对高温、强酸强碱、紫外线和过氧化氢酶均具有较好的稳定性，而胃蛋白酶和胰蛋白酶会使其失活。     

鲎素Tachyplesin Ⅰ在大肠杆菌中的重组表达及其抑菌活性

为了高效制备抗菌肽,本文将鲎素抗菌肽目的基因Tachyplesin-1(TP1)在大肠杆菌BL21中进行表达。首先构建表达质粒pET32a-TP1并转化大肠杆菌BL21(DE3),在IPTG诱导下进行目的融合蛋白表达,并利用His标签与镍柱亲和层析对融合蛋白进行纯化。纯化后的融合蛋白经羟胺裂解液切割和质谱分析后,获得单一的TP1重组蛋白,最后对其抑菌活性进行表征。结果表明,重组菌在37℃经IPTG诱导后,融合蛋白表达成功,TrxA-TP1融合蛋白的分子量在20 kDa左右。经羟胺裂解得到的重组蛋白TP1对于金黄色葡萄球菌(Staphylococcus aureus)与枯草芽孢杆菌(Bacillus subtilis)均具有良好抑菌活性,最小抑菌浓度分别为6和10 mg/L。本研究为鲎素抗菌肽TP1的开发应用和大量生产奠定了基础。     

片球菌素BM-1免疫蛋白对甘露糖磷酸转移酶受体识别特异性研究

利用牛津杯法筛选到对片球菌素BM-1敏感的植物乳杆菌WQ0815和肠膜明串珠菌05-43。并利用Nisin诱导表达系统,在乳酸乳球菌NZ9000中诱导表达两敏感菌株的甘露糖磷酸转移酶系统IICD组分,抑菌实验表明不同来源的IICD组分皆可被片球菌素BM-1识别。进一步将片球菌素BM-1同源免疫蛋白分别与两种IICD组分同时在乳酸乳球菌中表达,抑菌实验表明:免疫蛋白可识别植物乳杆菌来源的IICD组分,不能识别肠膜明串珠菌来源的IICD组分。研究结果为IIa类细菌素的同源免疫蛋白对受体存在特异性识别提供了直接证据。     

cA-GFP融合蛋白在大肠杆菌中的高效功能性表达

为了实现乳酸乳球菌锚定蛋白cA在大肠杆菌中可溶表达以及直观检测其生物学活性,以乳酸乳球菌MG1363为模板,扩增N-乙酰葡萄糖胺糖苷酶AcmA基因,将其C末端序列与GFP基因融合,连接至大肠杆菌表达载体pET28a,构建重组质粒pET28a-cA-GFP,转化至大肠杆菌表达菌株BL21(DE3),通过低温诱导表达重组蛋白cA-GFP。工程菌超声破碎后的上清液与经热酸处理的乳酸乳球菌常温孵育,经SDS-PAGE和荧光显微镜检测。结果表明,工程菌可以表达分子量约53ku的目的融合蛋白质,与预期大小相符。cA-GFP通过锚定蛋白cA的引导回向锚定,成功将GFP展示在乳酸乳球菌表面,目的蛋白cA-GFP在乳酸乳球菌表面展示量为121mg/g干重菌体。GFP锚定至乳酸乳球菌后于4℃保存,连续6d测定其荧光强度,荧光强度仍可达82.2%,证明其稳定性较好。成功获得了具有生物学功能的cA-GFP可溶性蛋白,为进一步展示功能性外源蛋白奠定了坚实的基础。     

乳源血管紧张素转移酶抑制肽在乳酸乳球菌中的表达

在乳酸乳球菌中表达乳源血管紧张素转移酶抑制肽。选取了4种不同的来源于牛酪蛋白的血管紧张素转移酶(ACE)抑制肽,为了确保能够在人体消化液作用下正常发挥它们的ACE抑制活性,4种短肽以串联多肽(TP)的形式进行表达,并在各短肽单体间引入了人体内主要消化酶的酶切位点。根据TP的氨基酸序列和乳酸乳球菌的偏爱密码子,人工合成TP基因。然后将TP基因和绿色荧光蛋白(GFP)基因串联于载体pSEC-E7,从而构建了pSEC-TP:GFP质粒,实现了2种蛋白在乳酸乳球菌中的共表达。经电击转化,将该重组质粒转入乳酸乳球菌NZ9000中,获得重组菌株NZ9000(pSEC-TP:GFP)。用Nisin诱导TP:GFP蛋白表达。RT-PCR、激光共聚焦扫描显微镜和SDS-PAGE鉴定表达产物。RT-PCR结果表明,TP:GFP蛋白在RNA水平表达成功;SDS-PAGE表明目标产物是35 ku的条带。在乳酸乳球菌中实现了乳源血管紧张素转移酶抑制肽的表达。     

植物乳杆菌细菌素基因plnEF的克隆与异源表达

目的:构建目的基因plnEF原核表达系统,诱导工程菌BL21-p ET28a-PL-plnEF表达目的融合蛋白并纯化,以期获得大量纯度较高的植物乳杆菌PlnEF细菌素,开发新的食品防腐剂。方法:构建含plnEF基因的重组质粒,将其转入大肠杆菌BL21中,经过IPTG诱导大量表达目标蛋白,融合蛋白PlnEF经纯化后进行分子量大小、抑菌活性等的检测。结果:重组质粒pET28a-PL-plnEF在大肠杆菌BL21中成功表达,合成PlnEF蛋白,其分子量为15.6 k Da,且该融合蛋白对大肠杆菌JM109具有良好的抑菌活性。结论:plnEF基因片段能够在原核细胞中正确表达且具有活性,本文为进一步研究开发该细菌素作为生物防腐剂奠定了基础。     

硫氧还蛋白促进人胰岛素样生长因子-1在大肠杆菌中高效可溶表达

为实现人胰岛素样生长因子-1(Insulin-like growth factor-1,IGF-1)在大肠杆菌中的高效可溶表达,获得大量具生物活性的IGF-1。应用融合PCR技术构建trx-igf1融合基因,克隆至p ET30a载体。重组载体pET30a-Trx-IGF-1转化大肠杆菌C43(DE3),并进行条件优化大量表达可溶性蛋白Trx-IGF-1。利用His标签纯化表达产物,对纯化蛋白进行Western blot与生物活性分析。构建的pET30a-Trx-IGF-1重组载体转化大肠杆菌C43(DE3)后,在30℃培养条件下1 mmol/L IPTG诱导表达5 h,获得大量以可溶形式表达的融合蛋白Trx-IGF-1;采用Ni离子亲和层析,获得纯度达90%以上的融合蛋白,经Western blot检测具有IGF-1抗原活性。生物学活性检测显示,融合蛋白Trx-IGF-1能明显促进NIH3T3细胞增殖及细胞周期进展。本研究应用的载体蛋白Trx,能实现在大肠杆菌中高效可溶表达具生物活性的IGF-1,为包涵体蛋白在大肠杆菌中的可溶性表达提供借鉴。     

乳酸菌细菌素Avicin A的高效表达及抑菌活性研究

乳酸菌细菌素是一种广谱抑菌素,已成为天然食品防腐剂研究与开发的热点。文章进行了该类细菌素在大肠杆菌中的异源表达,并应用Western-blot鉴定。利用组氨酸标签纯化及Trx-Tag标签的切除,对重组蛋白的抑菌活性进行检测。根据GenBank数据库中公布的细菌素Avicin A基因设计引物,以本实验室分离自酸马乳中的乳酸菌XM-38株为模板,采用PCR方法扩增了细菌素Avicin A片段,利用原核表达载体pET-32a(+)构建重组质粒HIS-Trx-Avicin A,将重组质粒转入大肠杆菌BL21(DE3)中表达,获得了可溶性表达的融合蛋白(HIS-Trx-Avicin A)。SDS-PAGE分析HISTrx-Avicin A蛋白大小约为42 ku,表达量约占菌体总蛋白的19%。经亲和柱层析纯化后,得到纯化的HIS-Trx-Avicin A蛋白,含量180μg/mL。用肠激酶对融合蛋白HIS-Trx-Avicin A进行解离,获得浓度为20μg/mL的目的蛋白Avicin A。对HIS-Trx-Avicin A进行Western-blot鉴定,表明该细菌素蛋白得到了重组表达,对多种菌的抑菌实验表明,该重组蛋白具有良好的抑菌活性,研究的Avicin A为食品添加防腐剂提供了一个切实可行的来源。     

广谱抗菌肽——片球菌素pediocin PA-1

片球菌素PA-1是一种广谱的乳酸菌细菌素,它对食品工业中的腐败菌单核细胞增生李斯特氏菌有强烈的抑制作用,是Ⅱa类细菌素中研究最深入的一种抗菌肽,具有很好的作为食品生物防腐剂开发的应用前景。对近年来关于片球菌素PA-1的结构、作用方式及在食品中的应用作一综述,并对其未来的应用前景,在蛋白质工程、基因工程和化学合成方面进行性质改进做出了展望。     

乳酸片球菌素二级结构与抑菌活性的关系

通过圆二色光谱法对乳酸片球菌素的二级结构进行研究,发现乳酸片球菌素的α-螺旋含量约为6.8%,β-折叠含量约为20%,转角所占比例为20%。当乳酸片球菌素的α-螺旋含量增加,β-折叠、转角含量降低时,细菌素活性随之降低,在pH6.0的低盐溶液中,乳酸片球菌素的结构稳定,此时抑菌活性最高;在pH12.0或者高浓度的盐溶液中,乳酸片球菌素α-螺旋含量增加至58%,β-折叠结构消失,细菌素活性丧失。     

Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1

Pediocin PA-1 is a representative class IIa bacteriocin which is small and heat-stable and has a consensus motif, -YGNGV-. The plasmid pQE40PED, encoding pediocin PA-1 fused with His-tagged mouse dihydrofolate reductase (DHFR), was constructed and introduced into Escherichia coli M15 strain. The fusion protein was overexpressed in the strain after induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active pediocin PA-1, the purified fusion protein was cleaved by Factor Xa protease and the liberated pediocin PA-1 was finally purified by ultrafiltration with a 75% yield. The molecular mass of the purified recombinant pediocin PA-1 was the same as that of native pediocin PA-1 on an electrophoresis gel.     

Pediocin PA-1, a wide-spectrum bacteriocin from lactic acid bacteria

Pediocin PA-1 is a broad-spectrum lactic acid bacteria bacteriocin that shows a particularly strong activity against Listeria monocytogenes, a foodborne pathogen of special concern among the food industries. This antimicrobial peptide is the most extensively studied class Ila (or pediocin family) bacteriocin, and it has been sufficiently well characterized to be used as a food biopreservative. This review focuses on the progress that have been made in the elucidation of its structure, mode of action, and biosynthesis, and includes an overview of its applications in food systems. The aspects that need further research are also addressed. In the future, protein engineering, genetic engineering and/or chemical synthesis may lead to the development of new antimicrobial peptides with improved properties, based on some features of the pediocin PA-1 molecule.     

Detection of pediocin PA-1-producing pediococci by rapid molecular biology techniques

Five bacteriocinogenic lactic acid bacteria strains (347, X13, Z102, A172 and P20) independently isolated from fermented sausages were identified asPediococcus acidilacticiby carbohydrate fermentation patterns and other biochemical characteristics. This fact, together with their activity againstListeria monocytogenes, suggested that they could be pediocin PA-1 producers. Rapid molecular biology techniques were used to detect the pediocin PA-1 operon in these strains. PCR, dot-blot and Southern hybridization, and DNA sequencing results confirmed that all of them had the genetic determinants for pediocin PA-1 biosynthesis encoded in a 9.4 kb plasmid. The bacteriocin produced byP. acidilactici347 has been purified by a procedure that included ammonium sulphate precipitation and cation exchange, hydrophobic-interaction and reverse-phase chromatography. The amino acid sequence of this bacteriocin was identical to pediocin PA-1, confirming the expression of the pediocin PA-1 genes.     

片球菌素的纯化及稳定性研究

片球菌素是一类具有良好热稳定性的未修饰的小分子蛋白质。其理化性质稳定,能抑制多种革兰氏阳性菌,对单核细胞增多症李氏杆菌抑制作用最为显著。文中着重总结了近年来在片球菌素稳定性及分离纯化方法领域的研究进展,同时也对其研究过程中存在的问题进行了探讨。     

河南华溪蟹金属硫蛋白分泌型表达载体的构建、表达及纯化

目的：构建河南华溪蟹金属硫蛋白（metallothionein,MT）分泌型表达载体并诱导其可溶表达。方法：采用基因重组技术,将河南华溪蟹MT基因亚克隆至碱性磷酸盐启动子（alkaline phosphatase promoter,phoA）分泌型原核表达载体,转化大肠杆菌BL21（DE3）,通过低磷酸盐诱导重组蛋白表达,经Ni2+螯合柱分离纯化后,利用紫外光谱扫描分析、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gelelectrophoresis,SDS-PAGE）及Western blotting检测鉴定重组MT。结果：phoA-MT分泌型表达载体构建成功,工程菌经低磷酸盐诱导后重组MT以可溶形式获得表达,紫外光谱扫描和Western blotting证实表达产物的正确性,SDS-PAGE分析其纯度较高,主要以单体和二聚体的形式存在,其分子质量分别约为7.5、15 kD。结论：成功实现了河南华溪蟹MT的重组表达。     

Pediocin PA-1 production during repeated-cycle batch culture of immobilized Pediococcus acidilactici UL5 cells

Pediocin PA-1 production by Pediococcus acidilactici UL5 cells immobilized in kappa-carrageenan/locust bean gum gel beads was studied during repeated-cycle batch (RCB) culture with pH control in Man Rogosa and Sharpe (MRS) broth supplemented with 1% glucose and whey permeate (SWP) medium. The pediocin PA-1 production by free P. acidilactici cells pH-controlled batch culture has reached 2048 and 4096 AU ml(-1) after 11 and 12 h of incubation, with volumetric productivities of 187 and 342 AU ml(-1) h(-1) in SWP and MRS media, respectively. In RCB culture, immobilized cells reached a maximum concentration of 7.3+/-0.2 x 10(10) and 4.3+/-0.9 x 10(10) cfu g(-1) of beads in MRS and SWP media, respectively. The maximum pediocin PA-1 activity obtained during RCB fermentation was 4096 AU ml(-1); it was attained after only 0.75 and 2 h of incubation in MRS and SWP media, respectively. The corresponding volumetric productivities were 5461 and 2048 AU ml(-1) h(-1). Pediocin PA-1 production in the RCB culture was highly stable over 12 fermentation cycles carried out over 3 d in SWP media.     

Bacteriocins Applied to Food Packaging Materials to Inhibit Listeria monocytogenes on Meats

Bacteriocins in powders were produced from milk-based media and applied to food packaging films. Nisin and pediocin “powders” were retained in casings during dialysis. Antilisterial casings were prepared by internal coating with pediocin. Antilisterial activity applied in powdered form was retained during processing and retained on contact food packaging surfaces. Pediocin powder was applied to plastic packaging bags at 7.75 μg/cm². Meats and poultry samples were inoculated with Listeria monocytogenes. The bags coated with pediocin powder completely inhibited growth of inoculated L. monocytogenes through 12 wk storage at 4°C. Applying bacteriocins to food packaging films is an effective approach to reduce L. monocytogenes contamination in meats and poultry.     

Characterization and heterologous expression of a class IIa bacteriocin,plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.     

Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria

The present work compares, under the same stated experimental conditions, the antimicrobial activity of crude and purified enterocin L50, pediocin PA-1, nisin A and lactocin S, produced by lactic acid bacteria (LAB) isolated from Spanish dry-fermented sausages. The bacteriocins were purified to homogeneity by ammonium sulphate precipitation, gel filtration (for lactocin S), and cation-exchange, hydrophobic-interaction, and reverse-phase-chromatography; high yields of pure bacteriocins were obtained. Minimal inhibitory concentration (MIC) of pure enterocin L50, pediocin PA-1, nisin A and lactocin S was determined against a broad spectrum of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The purified bacteriocins showed a broad antimicrobial spectrum similar to that exerted by crude bacteriocins. Enterocin L50 and pediocin PA-1 were very active againstListeria monocytogenes, which was quite resistant to nisin A and lactocin S. Enterocin L50 also displayed antimicrobial activity againstStaphylococcus aureus,Clostridium perfringensandClostridium botulinum. However, these pathogens were weakly inhibited, or not at all, by the other pure bacteriocins.