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《生物技术通报》2017,(5)
鰤鱼诺卡氏菌(Nocardia seriolae)是鱼类诺卡氏菌病的主要病原,为兼性胞内菌。生物信息学分析显示鰤鱼诺卡氏菌DmpA基因的表达产物为分泌蛋白,且可能定位于宿主细胞的线粒体上。通过构建重组质粒p EGFP-DmpA和pc DNA-DmpA、鰤鱼诺卡氏菌胞外产物鉴定、亚细胞定位、过表达等方法,对鰤鱼诺卡氏菌DmpA基因进行了克隆、亚细胞定位及初步功能研究。结果表明在鰤鱼诺卡氏菌胞外产物中鉴定到了DmpA蛋白,证实其为分泌蛋白。亚细胞定位研究发现DmpA-GFP融合蛋白均匀地分布在FHM细胞中,与线粒体分布不重合,说明DmpA蛋白并不定位在线粒体上。DmpA-GFP融合蛋白的表达会改变FHM细胞线粒体的分布为团块状。DmpA在细胞中的过量表达对细胞核无明显影响,表明该基因无诱导细胞凋亡的功能。通过对鰤鱼诺卡氏菌DmpA的克隆、亚细胞定位和过表达研究,为进一步研究该基因的功能和深入了解鰤鱼诺卡氏菌的致病机理奠定了基础。 相似文献
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【背景】鰤诺卡氏菌是一种典型的条件致病菌,感染鳢、鲈等多种名优鱼类,易造成存在机体损伤或免疫机能下降的鱼持续性感染,给水产养殖业造成了巨大损失。【目的】了解临床分离鳢源鰤诺卡氏菌对乌斑杂交鳢(斑鳢♀×乌鳢♂)的致病性,并从全基因组层面了解该病原菌的基因组和致病因子信息,为鰤诺卡氏菌后续病原学及鰤诺卡氏菌病防治技术和疫苗的开发研究提供有利的数据支撑。【方法】鰤诺卡氏菌NK201610020通过回归感染试验和发病鱼靶器官组织病理分析,了解鰤诺卡氏菌的毒力和病理特征。通过对试验菌进行全基因组测序和比较基因组学分析,挖掘该菌的基因组与毒力特征。【结果】回归感染试验结果显示,除1.5×103组外,其余5个感染组致死率高达90%,LD50为1.079×103 CFU/mL,说明试验菌毒力较强。组织病理学观察到肝、脾、肾呈现严重的病理损伤,而且有肉芽肿结构形成。试验菌全基因组测序发现,全基因组大小为8294329bp,GC含量为68.10%,共预测到编码基因7812个。比较基因组学分析发现,不同地区不同宿主来源鰤诺卡氏菌在基因组基础特... 相似文献
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鱼类的诺卡氏菌病主要由鰤鱼诺卡氏菌(Nocardia seriolae)引起。为探讨鰤鱼诺卡氏菌的毒力因子及其感染致病机制,本研究通过对鰤鱼诺卡氏菌全基因组序列的分析,发现了一个编码超氧化物歧化酶(superoxide dismutase,SOD)的基因。生物信息学分析显示该基因编码一个分泌蛋白,可能是鰤鱼诺卡氏菌潜在的毒力因子。本研究通过基因克隆获取了鰤鱼诺卡氏菌SOD基因(NsSOD),其长度为624 bp。利用双酶切技术在含有GST标签的原核表达载体pGEX-6P-1中插入NsOD基因片段,成功构建了其重组表达载体并命名为pGEX-SOD。将pGEX-SOD导入大肠杆菌BL21(DE3)感受态细胞,获得了原核表达菌株BL21/pGEXSOD并对其诱导表达条件进行了摸索,确定25℃、IPTG(1 mmol/L)诱导14 h,可成功获得具有生物活性的可溶性NsSOD重组蛋白。随后利用GST标签亲和柱纯化NsSOD重组蛋白,并进行酶活性质测定,确定NsSOD重组蛋白的活性为2.76酶活力单位。通过对NsSOD进行基因克隆、原核表达、重组蛋白纯化及体外酶活性质测定,为进一步研究NsSOD的功能和深入了解鰤鱼诺卡氏菌的致病机理奠定了基础。 相似文献
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【背景】鱼类诺卡氏菌病潜伏期和病程较长,感染率和死亡率较高,给水产养殖业带来较大的经济损失,其病原鰤诺卡氏菌(Nocardia seriolae)是胞内寄生菌,侵入细胞后引起慢性感染是主要的致病机制。【目的】构建鰤诺卡氏菌侵染大口黑鲈(Micropterus salmoides)头肾巨噬细胞体外模型,观察鰤诺卡氏菌侵染巨噬细胞的过程并探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【方法】采用密度梯度离心法分离巨噬细胞,通过特异性染色和PCR扩增巨噬细胞表达基因mpeg1对细胞进行鉴定,并通过CCK-8法和氧呼吸暴发活性测定检测巨噬细胞的活性;通过倒置荧光显微镜和流式细胞术观察侵染过程中细菌与细胞的形态与数量变化;通过双荧光流式细胞术检测、乳酸脱氢酶(lactate dehydrogenase, LDH)释放试验及线粒体膜电位检测,探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【结果】从大口黑鲈头肾分离获得纯度高的巨噬细胞,经染色和PCR法鉴定为巨噬细胞;筛选出最优的体外培养条件为1640培养基+1%青霉素链霉素+1%胎牛血清。在脂多糖刺激后,巨噬细胞的氧呼吸暴发能力显著提高(P<0.05)。GFP-鰤诺卡氏菌侵染细胞2 h后细菌被细胞吞噬,4 h细胞变圆且贴壁率降低,6 h细菌大量繁殖并包围细胞,8 h后细胞大量死亡。凋亡相关实验结果表明,侵染初期巨噬细胞凋亡率增加,LDH释放增加,线粒体膜电位下降;随着侵染时间延长,细胞凋亡率下降、LDH释放量及线粒体膜电位下降减少,说明鰤诺卡氏菌对巨噬细胞起先促进后抑制凋亡的作用。【结论】通过密度梯度离心法成功分离大口黑鲈头肾巨噬细胞,并通过鰤诺卡氏菌侵染细胞后初步摸清鰤诺卡细菌在细胞水平的致病机理,建立了鰤诺卡氏菌侵染大口黑鲈头肾巨噬细胞的体外模型;证实了鰤诺卡氏菌可侵染巨噬细胞并抑制细胞凋亡,从而达到在巨噬细胞内存活,为进一步开展鰤诺卡氏菌与巨噬细胞相互作用并阐明鰤诺卡氏菌的致病机制奠定了研究基础。 相似文献
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养殖乌鳢诺卡氏菌病及其病原研究 总被引:9,自引:0,他引:9
2006年6月至7月,浙江萧山的部分乌鳢养殖池塘出现大规模死鱼现象,发病率达35%,死亡率100%。病死鱼鱼龄多在18个月左右,体长30-35 cm。症状主要表现为体表、鳍条充血,肝、脾、肾等内脏器官出现直径1-5 mm的乳白色结节。从病鱼的肝脏和肾脏中分离到菌株W060622,经光镜、电镜观察,在形态上均表现为长或短的分支状杆菌。回归感染试验证实W060622即引起此次乌鳢结节病的病原菌。通过16S rRNA基因的全序列测定,并与Genbank中相关序列进行比对,发现W060622与鰤鱼诺卡氏菌Nocardia seriolea JCM3360T (Z36925)的16S rRNA基因序列相似性达99.9%。结合菌株回归感染、形态学和生物学特征及16S rRNA分子鉴定,确定W060622为鰤鱼诺卡氏菌。 相似文献
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[背景] 鰤鱼诺卡氏菌(Nocardia seriolae)是一种严重危害水产养殖业的病原菌,可引起以体表溃疡、出血及组织器官形成结节为特征的鱼类慢性肉芽肿疾病,目前尚无有效的防治方法。[目的] 明确引起安徽省临泉县某养殖场加州鲈(Micropterus salmonoides)结节病的病原菌,探讨其致病性,为该病的有效防治提供科学依据。[方法] 取肝脏结节病灶接种于TSB培养基分离优势细菌,利用表型检查结合分子生物学方法鉴定分离菌株。进一步通过检测分离菌株的毒力基因、测定其对加州鲈的半数致死量(LD50)以及所感染加州鲈的组织病理学变化与组织载菌量,分析其致病性。[结果] 从病鱼体内分离到一株优势菌株NI,综合NI分离株的表型特性、16S rRNA基因序列与鰤鱼诺卡氏菌参考株相应序列的一致性以及特异性PCR扩增结果,确定其为鰤鱼诺卡氏菌。鰤鱼诺卡氏菌NI分离株携带毒力基因gapA、ibeA和mip,人工回归感染后加州鲈出现与自然病例相似的症状,其对加州鲈的LD50为2.58×106 CFU/尾。组织病理学观察到头肾、心脏、肝脏、胃和脾脏均出现慢性肉芽肿病变,肠管肌层疏松、肠绒毛脱落,肌肉组织中肌纤维疏松、间隙增宽。qPCR检测结果显示,组织中鰤鱼诺卡氏菌载量由高到低依次为头肾、心、肝、胃、脾、肠和肌肉。[结论] 鰤鱼诺卡氏菌是引起此次加州鲈结节病的病原菌,对该菌致病性的研究为加州鲈诺卡氏菌病的防控提供了理论依据。 相似文献
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为对诺卡氏菌病进行快速早期诊断,建立了鰤鱼诺卡氏菌(Nocardia seriolae)TaqMan qPCR检测方法。以鰤鱼诺卡氏菌的管家基因SecA为靶标设计引物和探针,对反应条件进行优化,制作标准曲线,进行灵敏性、重复性和特异性测试,并将建立的方法应用于临床样品的检测。结果表明,优化后,引物和探针终浓度分别为0.3和0.1μmol/L,退火延伸温度60℃时,qPCR在9.85×1010—9.85×100 copies内呈良好的线性关系,灵敏度最高达9.85 copies;重复性实验结果显示,组间和组内变异系数均小于1%;特异性结果表明,对无乳链球菌、弗氏柠檬酸杆菌和维氏气单胞菌等13种病菌均无扩增曲线;临床对42份大口黑鲈样品进行检测,结果表明,qPCR检出率比普通PCR提高14.24%;对发病大口黑鲈的组织及结节部位检测显示,结节部位菌载量大幅高于非结节部位,最高相差1000倍;SecA基因分析结果显示,SecA基因在传代中和种内高度保守,种间具有一定的离散性,是个很好的用于诺卡氏菌种鉴定的靶基因。研究建立的鰤鱼诺卡氏菌qPCR检测方... 相似文献
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为了探讨熊本牡蛎(Crassostrea sikamea)在长期饥饿胁迫下的生理代谢响应,测定了长期饥饿(0~80 d)以及再投喂(90~118 d)状态下熊本牡蛎的耗氧率、排氨率以及肝胰腺组织乳酸脱氢酶、琥珀酸脱氢酶、丙酮酸激酶、超氧化物歧化酶、碱性磷酸酶和溶菌酶的活性。结果表明:长期饥饿下,熊本牡蛎的耗氧率和排氨率极显著降低(P<0.01);随着饥饿时间的延长,熊本牡蛎乳酸脱氢酶、琥珀酸脱氢酶、丙酮酸激酶、超氧化物歧化酶和碱性磷酸酶活性均呈现波动下降的趋势,溶菌酶活性呈现波动上升的趋势;再投喂后熊本牡蛎的耗氧率、排氨率均极显著升高(P<0.01);长期饥饿对熊本牡蛎代谢和免疫功能造成损伤,熊本牡蛎通过降低代谢率和代谢酶的活性应对长期饥饿胁迫。本研究可为海水贝类饥饿胁迫下代谢调节机制的研究提供参考。 相似文献
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【背景】鰤诺卡氏菌(Nocardia seriolae)是一种革兰氏阳性好氧菌,易引起鲈形目鱼类结节病发生,近几年对大口黑鲈、杂交鳢等名优鱼类的危害越来越大。【目的】比较分析9株不同年份、不同地区患结节病的不同淡水鱼品种分离的鰤诺卡氏菌的种间同源关系、生长特性、致病性以及药物敏感性差异,为进一步研究制定相应的防控与治疗措施提供基础数据和科学依据。【方法】在9株试验菌生理生化实验鉴定结果基础上,采用16SrRNA基因序列比对、限制性片段长度多样性比较菌株间的同源性;扩增看家基因secA1基因序列进行不同来源菌株种内相似度比对;通过体外培养比较不同菌株的生长情况;人工注射感染分析9株试验菌对大口黑鲈的致病性差异;采用微量肉汤二倍稀释法测试9株试验菌对13种抗菌药物的最小抑菌浓度;运用PCR技术检测菌株携带毒力基因和耐药基因的情况。【结果】生理生化结果显示9株试验菌与鰤诺卡氏菌的基本理化特征相符;9株试验菌聚类分析聚为一类,种内相似度高,限制性片段长度多样性酶切图谱相同,生长特性无明显差异;9株试验菌对大口黑鲈致病性无显著差异,但毒力基因携带情况略有不同,其均携带mce1A、mig和pup基因,而dop和whiB3只在部分菌株检出;9株试验菌均对氟甲喹和氨苄西林耐药,除NS1外,都对磺胺间甲氧嘧啶耐药,对大部分抗菌药物敏感,主要携带blaTEM和sul1耐药基因。【结论】不同时间、不同地区及不同宿主分离的9株鰤诺卡氏菌在生物学特性、致病性和药物敏感性等方面无明显差异;同源性相近,提示华南地区感染淡水鱼的鰤诺卡氏菌可能为同一流行株型。研究结果为鰤诺卡氏菌的发病机制及防控技术的进一步研究奠定了基础。 相似文献
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Eighteen components in rainbow trout serum were tested for variability among individuals and stability during storage. In addition, the precision of an automated serum analysis system was determined. Stability of serum components was observed over 42 days at temperatures of 25° C, 4° C and - 10° C. Components tested included: albumin, total protein, blood urea nitrogen, cholesterol, chloride, glucose, potassium, sodium, cholinesterase, alkaline phosphatase, lactic dehydrogenase, a-hydroxybutyrate dehydrogenase, glutamic pyruvic transaminase, phosphohexose isomerase, inorganic phosphorus, calcium, creatinine, and creatine phosphokinase. Fish serum was generally more stable than human serum when stored at 25° C and 4° C and similar in stability at - 10° C. Precision of analytical methodologies was excellent for all components measured except creatine phosphokinase. 相似文献
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扎龙自然保护区雌性丹顶鹤血液生化指标 总被引:1,自引:0,他引:1
2004年11月至2005年9月,采用自动生化分析仪对扎龙自然保护区10只雌性成体丹顶鹤在越冬期、繁殖期和迁徙期的20项血液生化指标(总蛋白、白蛋白、球蛋白、直接胆红素、间接胆红素、总胆红素、血糖、尿素氮、肌酐、总胆固醇、甘油三酯、谷草转氨酶、谷丙转氨酶、碱性磷酸酶、肌酸肌酶、羟丁酸脱氢酶、乳酸脱氢酶、钙离子、无机磷和镁离子)进行了分析.结果表明:除总蛋白、球蛋白和血糖外,研究区雌性丹顶鹤血液中的其他指标均在不同时期之间表现出显著(P<0.05)或极显著差异(P<0.01).说明总蛋白、球蛋白和血糖可作为雌性丹顶鹤血液生化指标的可靠参考值,其他17项血液生化指标的年变化动态体现出雌性丹顶鹤在不同生理时期的生理特征和生态适应性.如果以这17项血液生化指标作为雌性丹顶鹤血液生化指标的参考值,就必须考虑不同生理时期对其含量的影响. 相似文献
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Sera from 129 squirrel monkeys, Saimiri sciureus, were analyzed for 15 chemical constituents. The values obtained were then analyzed for statistical significance causing the following sets of variables: (1) colony-bred versus noncolony-bred, (2) karyotype (3) vendor, (4) sex and (5) dietary iodine supplementation versus nonsupplementation. Calcium, inorganic phosphorous, albumin, uric acid, blood urea nitrogen, glucose, alkaline phosphatase and potassium were high in colony-bred animals. Cholesterol, total protein and chloride were lower in colony-bred animals than in noncolony-bred animals. No differences were seen in total bilirubin, lactic dehydrogenase, serum glutamic oxaloacetic transaminase and sodium. When the noncolony-bred animals were separated by karyotype, total protein was higher and chloride was lower between animals from Peru versus from Guyana. Colombian animals had total protein values lower than Peruvian and lactic dehydrogenase values higher than Peruvian. Colony-bred Peruvian monkeys had serum glutamic oxaloacetic transaminase values higher than colony-bred Colombian monkeys. No differences were observed between monkeys from different vendors. Chemical constituents higher between noncolony-bred males and females were calcium and alkaline phosphatase. There were no differences observed for colony-bred males and females. Dietary iodine supplementation appeared to increase both total bilirubin and calcium. 相似文献
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猴头菇对小鼠抗疲劳作用的实验研究 总被引:19,自引:0,他引:19
分别以猴头菇干粉(猴头菇Ⅰ组)和猴头菇浸出液(猴头菇Ⅱ组)饲喂小鼠,观察猴头菇对小鼠血清乳酸脱氢酶(LDH)活力、血乳酸、血清尿素氮(BUN)、肝糖原、肌糖原含量及运动耐力的影响。结果表明:实验60d后,猴头菇Ⅰ、Ⅱ组LDH活力、肝糖原及肌糖原含量明显高于对照组(P<0.05或P<0.01);运动后血乳酸的水平和BUN的增量明显低于对照组(P<0.05或P<0.01);运动后血乳酸消除速率显著高于对照组(P<0.05);在运动耐力测定时在水中淹死的时间比对照组长得多(P<0.05)。提示:猴头菇具有明显的增强运动能力和解除疲劳的作用。 相似文献
15.
J M Morris G A Bubenik 《Comparative biochemistry and physiology. A, Comparative physiology》1983,74(1):21-28
1. Alkaline phosphatase (AP), cholesterol, creatinine, uric acid, total protein, albumin, bilirubin, urea nitrogen, calcium, glucose, lactic dehydrogenase (LDH) and serum glutamic-oxalacetic transaminase (SGOT) were measured in the plasma of three intact and three castrated male deer. 2. A statistically significant seasonal cycle of AP, cholesterol, creatinine and uric acid was found in intact but not in castrated animals. 3. Monthly levels of total protein, albumin, urea nitrogen, bilirubin and calcium were significantly higher in castrated deer. 4. On the other hand, monthly levels of LDH and SGOT were higher in intact animals. 相似文献
16.
乌鳢组织内三种同工酶的研究 总被引:6,自引:0,他引:6
本研究对乌鳢9种组织内的LDH、MDH和ATPase同工酶作了分析,结果表明,乌鳢各组织内的LDH、MDH有明显的组织特异性,LDH由两个基因编码。骨骼肌中的s-MDH也有两个基因编码。ATPase同工酶存在于乌鳢的多种组织中,其基因编码数有待进一步探讨。 相似文献
17.
Blood samples were collected from 118 white-tailed deer (Odocoileus virginianus) shot on the Fort Riley Military Installation in northeastern Kansas. Values for these deer for hematocrit, glucose, alkaline phosphatase, uric acid, total protein, albumin, and calcium were within the ranges reported in previous studies for undrugged white-tailed deer. Abnormally high concentrations of serum glutamic oxaloacetate transaminase (SGOT) and lactic dehydrogenase (LDH) were attributed to general trauma and tissue damage caused by shooting the deer. Fawns had higher concentrations of alkaline phosphatase than adults and had lower concentrations in winter than at other times of the year. Serum urea nitrogen (SUN) concentrations fluctuated seasonally. Elevated concentrations of SUN in adult males killed in December were attributed to an increased catabolism of muscle protein caused by low dietary intake and high energy requirements during the rut. Cholesterol concentrations varied seasonally without regard to age or sex. 相似文献
18.
重金属Cd、Zn、Cu、Pb对土壤微生物和酶活性的影响 总被引:3,自引:0,他引:3
采用室内培养实验(25℃),研究了不同培养时间下重金属Cd、Zn、Cu、Pb(浓度分别为50,800,400,800mg.kg-1)污染对土壤微生物和酶活性的影响。结果表明,土壤蔗糖酶、过氧化氢酶和脱氢酶活性随着培养时间的增加而显著下降,在培养20d的时候达到最小值,然后酶活性缓慢升高。Cu对脲酶活性以及Cd对酸性磷酸酶和脲酶活性的抑制作用随时间增加而增加。土壤微生物生物量碳、细菌、真菌和放线菌数量随培养时间的增加均表现出先降低后升高的变化趋势。Cd和Cu对微生物生物量氮的抑制作用则随着培养时间的增加而增强,在培养30d时微生物生物量氮到达最低值,分别较培养10天减少了12.6%和16.5%。 相似文献
19.
Serum lactic dehydrogenase, leucine aminopeptidase, 5-nucleotidase and alkaline phosphatase activities were investigated in a number of diseases involving the hepatobiliary system.Leucine aminopeptidase was found to be a sensitive indicator of biliary obstruction, serum 5-nucleotidase slightly less sensitive, and alkaline phosphatase appreciably less sensitive. Leucine aminopeptidase and 5-nucleotidase activities were often increased by malignant infiltration of the liver and primary hepatic disease even in the absence of jaundice.Serum lactic dehydrogenase was frequently increased in primary hepatic disease and malignant disorders but was not apparently affected by bile duct obstruction per se. Thirty-five of 45 patients with proved malignancy had increased lactic dehydrogenase levels.The highest leucine aminopeptidase levels were encountered in carcinoma of the head of the pancreas. The frequent increase in both serum lactic dehydrogenase and leucine aminopeptidase activities in patients with carcinoma of the head of the pancreas suggests that these combined estimations are useful laboratory procedures in the diagnosis of malignant extrahepatic obstruction. 相似文献
20.
Anthracycline antibiotics, such as doxorubicin and daunorubicin, constitute a group of wide spectrum therapeutic agents. Application of these drugs in chemotherapy is limited because of their toxic effects. Melatonin, the main secretory product of pineal gland, was recently found as a free radical scavenger and antioxidant.We decided to evaluate the tissue protective effect of melatonin against toxic effects of doxorubicin in six groups of rats. Rats were given doxorubicin (Dx) (45 mg/kg dose), melatonin (MEL) (10 mg/kg), first doxorubicin and then melatonin (DM), first melatonin and then doxorubicin (MD).The degree of kidney, lung, liver and brain cells' alterations were examined biochemically.In doxorubicin-treated group, malondialdehyde (MDA) levels of kidney, lung, liver and brain tissues were significantly increased but glutathione (GSH) levels were decreased compared to control rats. In the group in which first doxorubicin and then melatonin were given, MDA levels were significantly decreased compared to the doxorubicin-treated group.In doxorubicin-treated group, serum levels of creatinine, uric acid, blood urea nitrogen (BUN), Gamma-glutamyl transpeptidase (GGT) and Lactic acid dehydrogenase (LDH) were significantly increased while serum albumin and total protein levels were significantly decreased compared to control rats.Melatonin decreased the intensity of the changes produced by the administration of doxorubicin alone. Melatonin was quite efficient in reducing the formation of lipid peroxidation, restoring the tissue GSH contents and alterations of serum levels. 相似文献