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构建了含有荧光假单胞菌自身启动子PP303的中间质粒载体pGFP.然后利用mini-Tn5转座子,通过接合转移,将两个带有不同启动子的绿色荧光蛋白基因分别整合到荧光假单胞菌P303染色体上,获得了在488nm波长下发光稳定的P303m1和P303m3菌株.PCR鉴定和Southern印迹结果均证明绿色荧光蛋白已随机插入P303染色体.SDS-PAGE结果表明,含有PA1/04/03启动子的P303m3菌株GFP表达量低于含有PP303启动子的P303m1菌株,染色体标记的GFP表达量低于质粒标记.室内平板抑菌试验结果表明,P303m1与出发菌株P303抑菌活性相当,对九种植物病原真菌有较强的拮抗作用.定殖、生存竞争能力研究表明,荧光假单胞菌在自然土壤中和大白菜根际都具有较强的定殖能力.P303和P303m1在自然土壤中第60天的菌量分别为1.63×104和3.3×102cfu/g土(湿重),大白菜根际第50天的菌量分别为3.29×106和4.1×104cfu/g根(湿重). 相似文献
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为探究超级细菌NDM-1质粒水平传播的特性,以携带NDM-1质粒的猫源洛菲不动杆菌和人源醋酸钙不动杆菌为供体菌,以工程菌株大肠杆菌DH5α和大肠杆菌、沙门氏菌弱毒菌株为受体菌,通过接合试验验证了NDM-1基因通过质粒跨种传播的可能性。结果显示,猫源NDM-1质粒可以转入E.coli DH5α中,并可以DH5α为中介,转入猪霍乱沙门氏菌C500中;而人源NDM-1质粒仅转入E.coli DH5α中,未能转入其他菌株。NDM-1质粒在沙门氏菌C500中比在E.coli DH5α中较为稳定遗传。质粒的导入并不影响宿主菌的生长特性。本研究初步揭示了NDM-1的传播方式,表明NDM-1质粒能够跨越菌种界限,并可以大肠杆菌为中介,转移至其它菌株中。 相似文献
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应用异型双功能连接剂MBS(间-马来酰亚胺苯甲酸-N-羟基琥珀酰亚胺酯)将猪带绦虫六钩蚴18ku重组蛋白(TSOL18)和载体蛋白-牛血清白蛋白(BSA)进行连接,制备连接蛋白BSA-TSOL18,用以增强TSOL18的免疫原性.连接蛋白利用快速脱盐柱分离技术进行纯化;纯化后应用醋酸纤维薄膜电泳证明二者的结合情况;并用间接ELISA法检测不同摩尔比连接产物中抗原部分的活性.结果表明:TSOL18与BSA偶连摩尔比为1∶3时连接有效,且对抗原活性影响小,可满足进一步免疫的要求. 相似文献
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【背景】黏菌素是人医临床治疗多重耐药革兰阴性菌感染的“最后一道防线”药物,其同时作为饲料添加剂及治疗用药长期用于畜禽养殖业。2015年,我国研究人员首次报道了质粒介导的黏菌素耐药基因mcr-1,预示该道防线面临被攻破的风险。然而,杀菌浓度及亚抑菌浓度的黏菌素对mcr-1阳性质粒传播的影响仍未知。【目的】以流行最广的mcr-1阳性IncI2型质粒为研究对象,探究不同浓度黏菌素对该质粒接合转移频率的影响,为黏菌素的科学使用提供理论依据。【方法】使用肉汤法进行了不同浓度黏菌素（0.02-4 μg·mL-1）作用下携带mcr-1的IncI2型质粒的接合转移试验。通过实时荧光定量PCR（RT-qPCR）及构建的公式计算不同时间点（1-24 h）的接合转移频率,并分析不同浓度黏菌素对IncI2质粒接合转移频率的影响。使用PI染料、活性氧（ROS）检测试剂盒测定不同浓度黏菌素下供体菌和受体菌的细胞膜透过性和ROS产生量。选取了对照组与低中高浓度3个处理组（0.02、1、4 μg·mL-1）进行了转录组测序,使用Deseq2软件进行基因差异表达分析。采用Prism v8.2.0软件对不同组间接合转移频率、细胞膜透过性及ROS产生量的差异进行统计学分析。【结果】结合本研究建立的接合转移频率计算公式表明黏菌素浓度为4 μg·mL-1时可在不同时间点提高mcr-1阳性IncI2质粒3-10倍的接合转移频率,而其他浓度则无显著差异。转录组结果显示,与对照组相比,低中高浓度黏菌素均可显著提高IncI2质粒上IV型分泌系统相关基因的表达,其中包括virB1、virB2、virB5以及traC。此外,黏菌素增加了I型菌毛合成基因fim的转录水平。PI染色结果显示当黏菌素浓度为2和4 μg·mL-1时,可增强供体菌和受体菌的细胞膜透过性,且转录组结果也显示膜相关基因（ompAX、bamDE、lolB、yiaD、csgEF）的转录水平均出现显著上调。但是黏菌素作用后ROS产生量及相关基因的转录水平无显著提高。【结论】揭示了黏菌素可通过增加细菌IV型分泌系统活性、细胞膜透过性和菌毛的生成从而提高mcr-1阳性IncI2质粒在大肠杆菌间的接合转移频率。研究结果提示,杀菌浓度无法完全杀死mcr-1阳性大肠杆菌的同时,还能增加质粒传播速度。因此,畜禽养殖业中黏菌素的治疗使用可能维持mcr-1阳性质粒的存在及传播,此外鉴于黏菌素已批准用于人医临床,该现象有可能引起临床黏菌素治疗mcr-1阳性病原菌的失败。 相似文献
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A 6‐bp Deletion Variant in a Novel Canine Glutathione‐S‐Transferase Gene (GSTT5) Leads to Loss of Enzyme Function 
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下载免费PDF全文 S. Craft J. Ekena J. Sacco K. Luethcke L. Trepanier 《Journal of veterinary internal medicine / American College of Veterinary Internal Medicine》2017,31(6):1833-1840
Objectives
Glutathione‐S‐transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST‐theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs.Methods
Banked livers from 45 dogs of various breeds were screened for low GST‐theta activity by the substrate 1,2‐dichloro‐4‐nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole‐genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus.Results
Canine livers ranged 190‐fold in GST‐theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in Vmax for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over‐represented in Pembroke Welsh corgis (P < 0.0001) based on available whole‐genome sequence data.Conclusions
An Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST‐theta enzyme activity. This variant is significantly over‐represented in purpose‐bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens. 相似文献17.
Thomas Müller Peter Thanei Wolfgang Mücke Hans-Peter Kriemler Tammo Winkler 《Pest management science》1999,55(5):594-596
The metabolism of cyprodinil, a novel broad-spectrum fungicide, was investigated in rats. After single oral administration of 0.5 or 100 mg kg−1 body weight, [phenyl-U-14C]cyprodinil was rapidly eliminated, principally in the urine. The metabolite pattern in urine exhibited a significant sex-related difference with respect to the major metabolite. Males and females both produced a dihydroxy metabolite, N-4-(hydroxyphenyl)-4-cyclopropyl-5-hydroxy-6-methylpyrimidin-2-ylamine. Female rats conjugated this metabolite with sulfate exclusively at the 5-hydroxypyrimidinyl moiety, while males formed equal amounts of the monosulfate and a disulfate conjugate. The sex dimorphism in the conjugation reaction indicates the involvement of a sex-specific sulfotransferase that catalyzed the transfer of the second sulfate group. 相似文献
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以淀粉和乙酰水杨酰氯为原料,合成了淀粉支载的阿司匹林高分子前药,用核磁共振谱对其进行结构表征,用紫外分光光度计测定阿司匹林的接入率,并在人工胃液和人工肠液中进行体外释放研究．结果表明,阿司匹林成功接入到淀粉分子上,该复合物在不同介质的体外释放中均呈现零级缓慢释药且释药的开始阶段无爆释现象出现,与阿司匹林相比较,其作用时间明显增长,有望显著减轻药物对胃粘膜和肠的刺激作用． 相似文献
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Determination and transferability of plasmid‐mediated antibiotic resistance genes of the bacteria isolated from rainbow trout 下载免费PDF全文
下载免费PDF全文 Erol Capkin Saliha Ozdemir Rafet Cagri Ozturk Ilhan Altinok 《Aquaculture Research》2017,48(11):5561-5575
Antibiotic resistance and the presence of resistance genes (ARGs) were investigated in the bacteria isolated from rainbow trout (Oncorhynchus mykiss) from different trout farms located in Turkey. The most frequent types of antibiotic resistance were towards β‐lactams (cephalothin [70% of bacterial isolates], amoxicillin [63%], ampicillin [62%], ticarcillin [56%], aztreonam [51%]), macrolide [erythromycin, 68%] and sulphonamide [sulphamethoxazole, 51%]. Of bacterial isolates, 51% were multiple drug resistant (MDR), while 35% of the isolates were extensively drug resistant (XDR). None of isolates were pandrug resistant (PDR). The most common ARGs were ampC (36%) and sul1 (24%). The class 1 integron gene cassette was detected in 51% of the bacteria. There was a strong positive correlation between the antibiotic resistance rate and the presence of ARGs (r2 = .932). Gene encodes blaCTX‐M1, one of the extended spectrum beta‐lactamase enzymes, was first described in Aeromonas caviae, Photobacterium damselae, Pseudomonas luteola and Burkholderia cepacia. It was determined that 35% of the bacteria harboured at least one plasmid. Plasmid‐mediated ARGs were identified to be tetracyclines (tetA, tetB, tetC, tetD), sulphonamides (sul1, sul3) and β lactams (ampC, blapse). Thus, results suggest that ARG contamination situation deliberates resistance to tetracycline, aminoglycoside, chloramphenicol and sulphonamide. Therefore, the presence and activity of ARGs in fish and in environmental bacteria may play an important role in the spread of resistance genes among bacteria by transposition or integron gene cassettes. 相似文献
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从2014年和2016年保存的来自河南、广东、山东和湖北4个省1 100株食品动物肠道大肠杆菌中筛选出58株blaCTX-M-27阳性菌株,采用药敏试验测定产blaCTX-M-27的大肠杆菌对20种抗生素的敏感性,并用PCR的方法检测其所携带的其他耐药基因;通过脉冲场凝胶电泳分析各菌株之间的亲缘关系,通过接合或转化试验、复制子分型和Southern blot对携带blaCTX-M-27的质粒特征进行分析。结果显示,58株blaCTX-M-27阳性大肠杆菌对除了β-内酰胺类外的其他抗生素,尤其氟喹诺酮类呈现高水平耐药;大多数菌株还同时携带2~3种编码其他抗生素的耐药基因。PFGE分型结果表明,来源于同一采样地及不同省的菌株存在克隆现象,但不同省或来自于同一省份的不同采样地之间的菌株亲缘关系较远;菌株携带的blaCTX-M-27基因全部定位在质粒上,其中38株位于可接合的IncFIB质粒,另外20株位于不可分型的质粒。 相似文献