北方某水厂原水及处理过程中Ah受体效应的行为研究

以北方某城市给水厂的自来水为研究对象,采用重组基因酵母测定法对不同处理工艺单元出水的芳香烃受体(AhR)效应进行了检测.检测结果表明,原水具有明显的AhR效应经常规工艺处理后AhR效应不能得到很好的去除,而经臭氧-活性炭深度处理后,92%的AhR效应能够被有效去除.     

Functional expression of recombinant sweet-tasting protein brazzein by Escherichia coli and Bacillus licheniformis

Brazzein is an attractive sweetener candidate because of its sugar-like taste, high sweetness, and good stability at high temperature and wide pH range. This study was aimed to express and purify bioactive recombinant brazzein (rBrazzein). The rBrazzein gene was synthesized according to the preferred codons of Bacillus subtilis and successfully expressed in Escherichia coli and Bacillus licheniformis. In E. coli host, lower induction temperature of 30°C increased soluble rBrazzein (Ebrazzein) at high level. In B. licheniformis host, two signal peptides (Sec type and Tat type) were evaluated for the expression of rBarzzein in B. subtilis and B. licheniformis. However, only the Sec-type signal peptide guided the secretion expression of rBrazzein in B. licheniformis. The rBrazzein was expressed steadily and the highest yield reached about 57 mg/L at 36 h by small-scale fermentation. The purification procedure of rBrazzein by B. licheniformis (Bbrazzein) was thus established. Approximately 5 mg/L purified rBrazzein was obtained and the purity was 85%. The conformational state of rBrazzeins was confirmed by circular dichroism. The bioactivities of rBrazzeins were evaluated by sweet taste testing. The Bbrazzein and Ebrazzein were 266 times and 400 times sweeter than sucrose on a weight basis, respectively. The formation of disulfide bonds were both confirmed by LC/MS/MS and MALDI-TOF. The CD analysis indicated that Ebrazzein has a similar secondary structure with natural brazzein, which explained why Ebrazzein had a higher intensity of sweetness. This study demonstrated that B. licheniformis system is useful to produce active recombinant brazzein, and has potential food industry applications.     

一种特异性检测单增李斯特菌的质粒DNA标准物质

研制了一种质粒DNA标准物质,包含目前单增李斯特菌检测常用的靶基因序列——内化素基因(Inl A)序列,可适用于扩增Inl A基因的单增李斯特菌PCR相关检测方法。采用紫外分光光度法(UV)和高分辨电感耦合等离子体质谱(HR-ICP-MS)两种方法,多家实验室联合定值的方法,对该套标准物质的均匀性、稳定性、量值以及不确定度进行详细的考察,质粒DNA标准物质的最终定值结果为94.9(±5.1)ng/μL。该质粒DNA标准物质可用于微生物实验室对单增李斯特菌PCR相关检测进行检测结果质量控制、开展方法比对和能力验证等。     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

Genetic Carriers and Genomic Distribution of cadA6—A Novel Variant of a Cadmium Resistance Determinant Identified in Listeria spp.

Listeria monocytogenes is a pathogen responsible for severe cases of food poisoning. Listeria spp. strains occurring in soil and water environments may serve as a reservoir of resistance determinants for pathogenic L. monocytogenes strains. A large collection of Listeria spp. strains (155) isolated from natural, agricultural, and urban areas was screened for resistance to heavy metals and metalloids, and the presence of resistance determinants and extrachromosomal replicons. Of the tested strains, 35% were resistant to cadmium and 17% to arsenic. Sequence analysis of resistance plasmids isolated from strains of Listeria seeligeri and Listeria ivanovii, and the chromosome of L. seeligeri strain Sr73, identified a novel variant of the cadAC cadmium resistance efflux system, cadA6, that was functional in L. monocytogenes cells. The cadA6 cassette was detected in four Listeria species, including strains of L. monocytogenes, isolated from various countries and sources—environmental, food-associated, and clinical samples. This resistance cassette is harbored by four novel composite or non-composite transposons, which increases its potential for horizontal transmission. Since some cadAC cassettes may influence virulence and biofilm formation, it is important to monitor their presence in Listeria spp. strains inhabiting different environments.     

CAMBRIDGE PRIZE LECTURE DEVELOPING NEW STRAINS OF YEAST*

Genetic engineering or recombinant DNA technology is now routinely applied to the construction and development of new strains of brewing yeast. This is a direct consequence of the power of the technology which facilitates the modification, introduction and stable maintenance of specific genes in brewing yeast, without compromising the intrinsic brewing properties of the yeast itself. The way in which gene technology has been applied to the development of new strains of Bass yeast is briefly illustrated by the provision of plasmid-based systems for ensuring the stable maintenance of recombinant genes, the construction of amylolytic and β-glucanolytic yeast and the design and development of genetic systems for enhancing the value of waste brewers yeast. Commercial and regulatory issues are discussed.     

利用组成型表达载体pMG36e电转化德式乳杆菌保加利亚亚种的研究

以典型的乳酸菌组成型表达质粒pMG36e为载体,德式乳杆菌保加利亚亚种为受体.采用电转化方法研究受体菌株的生长状态与细胞壁处理方式、质粒浓度、电转化参数、复苏培养基组成与复苏培养时间、受体菌株的限制修饰作用对电转化效率的影响,探讨电转化的最适条件,以提高电转化效率.结果表明:选择含2.5%甘氨酸的SMRS培养基培养受体细胞至对数中期,收获制成感受态细胞,在电场强度12 kV/cm、电阻200Ω、电容25μF条件下电击转化,以含20 mmol/L MgCl2、CaCl2的SMRS培养基复苏培养2 h,涂板筛选得到较高的电转化效率.特别是采用受体菌株修饰的质粒进行电转化,转化效率提高30倍以上,达到6.3×104cfu/μg DNA.本研究结果为进一步构建乳酸菌组成型高效表达系统和食品级基因工程乳酸菌株提供试验依据.     

外源质粒DNA对小鼠肝脏基因表达谱的影响

研究了外源质粒DNA经胃肠道吸收可能对肝脏产生的作用机制。给Balb／c小鼠灌胃质粒pcDNA3 200μg，在灌胃后4h分离肝脏，提取肝脏的总RNA。利用寡核苷酸芯片对灌胃质粒pcDNA3后的Balb／c小鼠肝脏进行基因表达谱研究。结果发现17664个基因中，表达上调100条，表达下调41条。按基因功能分类，表达上调的基因可分为免疫应答基因、转录因子基因、信号转导基因、转运相关基因及代谢相关基因等；表达下调的基因主要为脂质代谢基因。灌胃外源质粒DNA后，肝脏组织主要表现为急性时相反应的加强、免疫反应的活化、细胞信号通路的活化以及脂质代谢途径的抑制。表明外源质粒DNA通过胃肠道途径可广泛调控肝脏的基因表达。     

扩张蛋白swollenin基因香菇表达载体构建及其初步转化

构建含有Swollenin基因(swo基因)的香菇表达载体,并通过PEG(polyethylene glyeol)介导的原生质体转化法实现对香菇的遗传转化,转化效率约为1个转化子/μg质粒DNA。PCR和杂交检测结果表明,swo基因已经整合到香菇的基因组中;栽培实验结果表明,两个转化子都能正常出菇,而且转化子Ⅱ表现出优良的生物学性状,将swo基因转化到香菇中对香菇新品种的培育进行探索。