Nisin生物合成相关基因分析

Nisin是乳酸乳球菌某些菌株产生的一种对细菌芽孢和多种革兰氏阳性菌有较好杀伤作用的小肽,广泛用于食品加工、医疗保健等诸多领域。合成Nisin的基因位于染色体上-可接合型蔗糖转座子,由三个启动nisA、nisR、nisF控制的nisA/ZBTCIPRKFEG构成,其中nisA因具有更强的启动强度,且表达诱导物和宿主菌均为食品级,方便、经济、安全,应用最为广泛。其他基因分别在Nisin合成、调控过程中起不同作用：自身保护性基因ninI、nisF、nisE、nisG的表达,使菌体获得对Nisin较好的耐受性;NisB和NisC与翻译后修饰有关;NisT可促进乳链菌肽前体转移至胞外;NisP则与nisin前体信号肽的切除有关。     

食品级高效诱导表达系统-NICE系统

乳酸菌NICE系统是在乳链菌肽诱导下由nisA启动子控制目的基因表达的,含nisR和nisK的两组分调节系统的高效诱导表达系统。由于NICE系统的诱导剂、宿主菌和载体都是食品级的,其应用前景十分广阔。     

通用真核质粒表达载体的构建

利用真核基因表达调控的原理,以pSV2-dhfr为起始材料,构建了两个通用的真核质粒表达载体pMAML1-dhfr和pMAML2-dhfr,它们包含人巨细胞病毒立即早期启动子/增强子调控元件,由两个转录方向相同或相反的表达单元组成.以荧火虫荧光素酶基因为报道基因,β-半乳苷酶基因为内对照,借助COS-7短暂表达系统,研究了它们对荧光素酶表达的影响,并比较了它们与出发载体pSV2-dhfr的相对强弱.     

乳链菌肽Nisin的生物合成及表达调控机制

乳链菌肽Nisin是乳酸乳球菌（Lactococcus lactis）产生的一种多肽类抗菌物质,是一种由34个氨基酸组成的羊毛硫细菌素。与Nisin合成有关的基因有11个,构成一个基因簇nisA（Z）BTCIPRKFEG。这些相关基因组成三个操纵子进行转录,分别是nisA（Z）BTCIP、nisRK和nisFEG。Nisin通过NisRK双组分调节系统诱导自身合成,而NisI和NisFEG赋予了Nisin产生菌对Nisin的免疫性。对于Nisin的生物合成机制人们展开了非常广泛的研究。本文对Nisin的结构、Nisin合成相关的基因簇、Nisin的生物合成及表达调控机制以及Nisin产生菌对Nisin的免疫性进行了综述。     

三角褐指藻(Phaeodactylum tricornutum)通用转化载体的构建

三角褐指藻(Phaeodactylum tricornutum)是开展微藻生物柴油研究的理想材料。克隆了内源fcp基因簇的多个调控序列(启动子、终止子),构建了包括fcpB启动子-bar基因-fcpA终止子、以及fcpA启动子-多克隆位点(MCS)-fcpA终止子两个表达盒的通用转化载体pfcpA-MCS/fcpB-Bar,其特征是以bar基因作为选择标记,MCS区方便插入一至多个目的基因。新载体可用于三角褐指藻的重组蛋白表达、或油脂代谢相关基因的功能验证和代谢调控研究。     

肝特异性表达HCV5＇NCR调控荧光素酶质粒的构建及表达

小鼠白蛋白是肝组织特异性表达的蛋白,这种特异性是由白蛋白启动子所介导的.以2235A-1质粒为模板,通过PCR扩增获得小鼠白蛋白启动子/增强子基因片段,用小鼠白蛋白启动子/增强子基因片段取代pHCV-neo4质粒(含HCV5＇NCR调控荧光素酶基因)的CMV启动子,构建了一种白蛋白启动子启动转录的HCV5＇NCR调控荧光素酶表达质粒(pA1b-HCV).该质粒能在小鼠肝癌细胞中表达且较小鼠其它癌细胞中表达水平明显增高,表明成功地构建了肝特异性表达的HCv5＇NCR调控荧光素酶表达质粒.该研究为建立肝特异性表达的HCV5＇NCR转基因小鼠模型奠定了基础,对评价HCV特异性反义药物及肝靶向性运载系统的作用具有重要的实际意义.     

双控表达载体系统的构建及其在基因表达方面的应用（英文）

可严格调控性是体现原核表达载体优越性的重要指标。构建了一种双控双调节原核表达载体系统,用双载体控制调节目的基因的表达,即SP6启动子(promoter)+乳糖(lac)调节基因表达系统和araB启动子(promoter)+ara C调节基因表达系统,分别由乳糖类似物IPTG和阿拉伯糖(L-arabinose)诱导目的基因的表达。该系统由2个表达载体共同完成目的基因的表达。pE SP-1为主表达载体,即目的基因克隆到此表达载体上,由SP6启动子(promoter)+乳糖(lac)调节基因调控;pA RA-SP6为辅助表达载体,该载体通过SP6 RNA聚合酶的表达来控制调节主表达载体的启动子(SP6),由araB启动子(promoter)+ara C调节基因调控。实验结果显示该双控双调节表达载体系统控制严格,并且表达蛋白的量具有可调控性。     

肝特异性表达HCV5′NCR调控荧光素酶质粒的构建及表达

小鼠白蛋白是肝组织特异性表达的蛋白 ,这种特异性是由白蛋白启动子所介导的 .以2 2 35A- 1质粒为模板 ,通过 PCR扩增获得小鼠白蛋白启动子 /增强子基因片段 ,用小鼠白蛋白启动子 /增强子基因片段取代 p HCV- neo4质粒 (含 HCV5′NCR调控荧光素酶基因 )的 CMV启动子 ,构建了一种白蛋白启动子启动转录的 HCV5′NCR调控荧光素酶表达质粒 (p A1 b- HCV) .该质粒能在小鼠肝癌细胞中表达且较小鼠其它癌细胞中表达水平明显增高 ,表明成功地构建了肝特异性表达的 HCV5′NCR调控荧光素酶表达质粒 .该研究为建立肝特异性表达的 HCV5′NCR转基因小鼠模型奠定了基础 ,对评价 HCV特异性反义药物及肝靶向性运载系统的作用具有重要的实际意义     

不同调控元件驱动下菊欧氏杆菌纤维素酶celY基因在大肠杆菌中表达的研究

目的:以菊欧氏杆菌(Erwinia chrysanthemi)基因组DNA为模板,通过PCR方法找到了该菌的β-1,4-内切葡聚糖酶celY基因及其调控元件并克隆至pUC19载体。为提高纤维素酶celY基因在原核细胞中的分泌表达量,比较了由不同启动子和信号肽调控的celY基因在大肠杆菌中的表达水平。方法:分别构建了由脂蛋白启动子、T7启动子、果胶酶信号肽调控的多种表达载体与由纤维素酶celY基因自身的启动子和信号肽调控的表达载体相比较。结果:由脂蛋白启动子、T7启动子、果胶酶信号肽调控的表达载体都不同程度提高了celY基因的分泌表达量。结论:脂蛋白启动子、T7启动子、果胶酶信号肽都不失为构建强分泌表达载体的可选元件。     

Microplate bioassay for nisin in foods,based on nisin-induced green fluorescent protein fluorescence

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.     

The nisin-controlled gene expression system: construction, application and improvements

Lactic acid bacteria are widely used in industrial fermentation. The potential use of these bacteria as homologous and heterologous protein expression hosts has been investigated extensively. The NIsin-Controlled gene Expression system (the NICE system) is an efficient and promising gene expression system based on the autoregulation mechanism of nisin biosynthesis in the Lactococcus lactis. In the NICE system, the membrane-located histidine kinase NisK senses the inducing signal nisin and autophosphorylates, then transfers phosphorous group to intracellular response regulator protein NisR which activates nisA promoter to express the downstream gene(s). The NICE system allows regulated overproduction of a variety of interest proteins by several Gram-positive bacteria, especially L. lactis. The essential elements for system construction, its application for expression of some biotechnologically important proteins and further improvements of this system are discussed.     

Bioassay for nisin in milk, processed cheese, salad dressings, canned tomatoes, and liquid egg products

A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp(uv) gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.     

Microplate Bioassay for Nisin in Foods, Based on Nisin-Induced Green Fluorescent Protein Fluorescence

A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 μg of nisin in cheese, and 1 μg of nisin per ml in salad dressings.     

Bioassay for Nisin in Milk, Processed Cheese, Salad Dressings, Canned Tomatoes, and Liquid Egg Products

A sensitive nisin quantification bioassay was constructed, based on Lactococcus lactis chromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variant gfp_uv gene under the control of the nisin-inducible nisA promoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.     

Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria: Comparison of Induction Level and Promoter Strength

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.     

Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3.

The lantibiotic nisin is produced by several strains of Lactococcus lactis. The complete gene cluster for nisin biosynthesis in L. lactis 6F3 comprises 15 kb of DNA. As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK. Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK. Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified. One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L. lactis 6F3 have now been identified. Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins. Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin. However, in comparison with the wild-type strain, these mutants were more sensitive to nisin. This indicates that besides nisI the newly identified genes are also involved in immunity to nisin. The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.