首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 643 毫秒
1.
目的探讨斑马鱼尿酸氧化酶基因的克隆与原核表达的方法,研究重组斑马鱼尿酸氧化酶(ZUO)的理化性质与生物学功能。方法用PCR的方法克隆斑马鱼尿酸氧化酶基因,用pET28a质粒进行原核表达。结果成功克隆了斑马鱼尿酸氧化酶基因并完成了测序鉴定,该基因在大肠杆菌表达系统中得到有效表达且具有体外氧化尿酸的生物学功能。目的蛋白为胞内可溶性蛋白,占可溶蛋白总量的50%左右。最佳诱导条件为27℃16 h,粗酶最佳溶解pH为9.16,在此缓冲液溶解条件下,粗酶比活性为1.94 U/mg。获得的重组ZUO产量达64.8 U/g湿菌,高于直接从动物肝脏组织萃取的尿酸氧化酶。结论斑马鱼尿酸酶基因能在大肠杆菌表达系统中表达,其溶解pH比基因工程表达的重组猪尿酸氧化酶略低,对后续开发痛风治疗新药有利。  相似文献   

2.
尿酸氧化酶在大肠杆菌中的表达、纯化及活性鉴定   总被引:1,自引:0,他引:1  
尿酸氧化酶(urate oxidase,Uricase,EC.1.7.3.3)是一种能将尿酸氧化为尿囊素的蛋白酶。合成黄曲霉(Aspergillus flavus)尿酸氧化酶基因,构建表达载体pET43.1a/uox,重组质粒经双酶切鉴定和序列分析,证明插入序列正确,转化到大肠杆菌(Escherichia coli)JM109,菌株经诱导表达尿酸氧化酶蛋白,目的蛋白经过超声破碎,经检测以可溶性蛋白为主;菌体经超声破碎后,上清经过阴离子柱和阳离子柱两步纯化,得到尿酸氧化酶纯品,纯品以分光光度法进行体外酶活性测定。结果显示:尿酸氧化酶在大肠杆菌中获得高效表达,目的蛋白占菌体总蛋白的50%;表达产物经过两步层析柱纯化,获得电泳扫描纯度为95%的纯品;在体外活性测定中具有分解尿酸的能力,在临床检测和治疗中有重要意义。  相似文献   

3.
以碱性蛋白酶生产菌克劳氏芽孢杆菌(Bacillus clausii)基因组DNA为模板PCR扩增获得尿酸氧化酶基因(BcU),插入原核表达载体pET28α中,构建表达载体pET-BcU,并转化大肠杆菌BL21(DE3)获得重组大肠杆菌BL21(DE3)/pET-BcU。经IPTG诱导,重组菌BL21(DE3)/pET-BcU表达出有活性的尿酸氧化酶,含空质粒的重组菌在同样条件下没有酶活。酶学性质分析显示,重组酶最适pH值为9.0,在pH值9.0~11范围内酶活几乎不变,是一种高碱性尿酸氧化酶。  相似文献   

4.
尿酸氧化酶(Urate oxidase,Uox)是一种催化尿酸氧化为尿囊素的酶,常用于尿酸的检测以及痛风和高尿酸血症治疗。文中从土壤中筛选出一株Uox高产菌株OUC-1,经16S rRNA部分基因序列分析,与苛求芽孢杆菌Bacillus fastidiosus序列相似度达99%。B.fastidiosus OUC-1的Uox经纯化后,分析表明该酶反应最适pH和温度分别为10.0和40℃;Uox以尿酸为底物反应动力学参数K_m值为(0.15±0.04)mmol/L(n=5)。Mg~(2+)能够提高该酶性活性,而Zn~(2+)和SDS能强烈抑制该酶的酶活。参考GenBank中苛求芽孢杆菌基因组中的uox基因序列,成功扩增出uox基因,通过SWISS-MODEL对Uox空间结构进行预测,推测该酶是同源四聚体,单亚基分子量为35.38 kDa。文中将uox基因克隆并在大肠杆菌中表达,为后续的Uox的性能改造提供条件和技术支持。  相似文献   

5.
目的:制备微囊化不可繁殖型尿酸氧化酶工程菌,以期研制一种降低血尿酸的口服药物,用于治疗高尿酸血症及痛风。方法:用甲醛灭活尿酸氧化酶工程菌,使其不可繁殖并保持酶活性;以壳聚糖和海藻酸钠为囊材,采用气流喷雾法制备微囊;用试剂盒体外评价微囊降解尿酸的效果。结果:经甲醛灭活后,工程菌繁殖活性丧失,其降解尿酸的能力降低85%;制备的微囊粒径呈正态分布于20~220μm,包封率达到99%以上;体外试验表明其具有降尿酸活性。结论:获得了具有降解尿酸活性的微囊化不可繁殖型工程菌。  相似文献   

6.
尿酸氧化酶基因的克隆、表达及其产物的应用   总被引:8,自引:0,他引:8  
克隆了产朊假丝酵母(Candida utilis)AS2-117尿酸氧化酶(Urate Oxidase,Uricase,EC1.7.3.3)的基因。将此基因插入原核表达质粒pET21a后转化大肠杆菌BL21(DE3),获得高表达的重组转化子菌株。经IPTG诱导,重组尿酸酶基因表达量可达菌体可溶性蛋白的40%。重组尿酸氧化酶为有酶活性的可溶蛋白。Western印迹分析证实表达产物有免疫学活性。经DEAE DE52纤维素离子交换柱层析纯化,目的蛋白纯度可达95%。重组蛋白和天然蛋白的理化特征比较证明重组蛋白的热稳定性有较大提高。酶盒配制和临床应用实验表明重组蛋白可代替天然蛋白进行临床血清尿酸的分析。  相似文献   

7.
尿酸氧化酶广泛用于常规临床分析和临床药物,在医学治疗和诊断中的重要性日益增加。为得到高产尿酸氧化酶的优良菌株,以大连黄海海泥、海水为材料,依次分别采用透明圈法、酶偶联分光光度法进行初筛和复筛,获一株高产尿酸氧化酶菌株Z7,根据菌株Z7的16S rDNA基因序列和形态学、生理生化特征,该菌株被鉴定为苛求芽孢杆菌(Bacillus fastidiosus)。酶学性质研究表明:尿酸氧化酶蛋白的分子量约为33.1 kD;最适作用温度是25℃,酶活高达673.7 U/mg;最适作用pH为8.0,在pH 8-9表现出较强的稳定性;Fe~(3+)、Ca~(2+)对尿酸氧化酶具有激活作用。Ag+、Hg~(2+)对该酶抑制性较强,使其几乎丧失活性。该菌株产酶活性及稳定性良好,可为尿酸氧化酶的工业化生产奠定理论基础,并具有潜在开发价值。  相似文献   

8.
为了研究微杆菌Microbacterium sp.ZZJ4-1菌株的耐热尿酸氧化酶(Uox)的性质,克隆其基因(uox),得到1个894 bp的开放阅读框。该基因与多数已报道的uox无明显同源性,仅与球形节杆菌Arthrobacterglobiformis的uox有72%的同源性。将基因插入质粒pET-15b构成pET-15b-uox表达载体,转化至Escherichiacoli BL21(DE3)中诱导表达。对重组Uox的主要理化性质研究表明:该酶由大小约为35 kDa的亚基组成;其最佳反应温度和pH分别为30℃和7.5;在65℃以下和pH 8.5~11.0范围内稳定;以尿酸为底物的Km值为0.22 mmol/L;Ag+、Zn2+、Cu2+和SDS均能完全抑制酶活,Tween 20、Tween 80和Triton X-100对酶活有一定的促进作用。该重组酶的耐热性是目前报道的重组Uox中最好的,这一特性有利于其在诊断治疗中的开发应用。  相似文献   

9.
目的:制备抗黄曲霉尿酸氧化酶单克隆抗体。方法:采用杂交瘤技术,获得2株针对重组黄曲霉尿酸氧化酶(rUOX)的杂交瘤细胞McAb17和McAb3;采用盐析和A蛋白亲合层析柱纯化该抗体。结果:McAb17和McAb3的腹水效价达到1∶512000,纯化后获得纯度大于95%的单抗,抗体亚类(型)分别为IgG1/k型和IgG2a/k型;ELISA结果显示制备的单抗与rUOX和Rasburicase(商品化rUOX)可发生特异反应。结论:制备了抗黄曲霉尿酸氧化酶单克隆抗体McAb17和McAb3,为检测rUOX在动物体内的代谢变化提供了良好的实验基础。  相似文献   

10.
目的 建立石英微孔板法酶底物检测尿酸酶活性的方法,并对方法进行验证.方法 摸索尿酸浓度与吸光度之间的线性关系,找到合适的范围,并验证方法的专属性、线性、重复性、中间精密度、准确性和耐用性.尿酸酶加入固定浓度尿酸溶液,酶促反应5 min后,通过酶标仪检测A292nm值变化计算尿酸的含量,根据单位时间内尿酸减少量计算出尿酸...  相似文献   

11.
This article describes a method for the determination of gasoline range, diesel range, and mineral oil range organics in soils and water. It represents the culmination of a series of efforts to go beyond typical GRO and DRO methodology currently available in the literature to include a quantitative determination of mineral oil organics having a boiling range up to C44. It also is the result of an attempt to develop a cost‐effective method that enables the analyst to quantify three different types of hydrocarbon components in one GC run under conditions without a concentration step. Method performance is comparable to that of current protocols for GRO and DRO determinations and validated further by comparisons to certified standards and in‐house standards. Accuracy as percentage recovery for GRO in water is 82 to 84 and 91 to 92% for soils. Accuracy as percentage recovery for MRO in water is 84 to 102 and 75 to 80% for soils. Accuracy as percentage recovery for DRO in water is 78 to 100 and 71 to 90% for soils. Results from the analysis of in‐house standards and certified standards for DRO and MRO gave higher recoveries than was demonstrated in the MDL studies. The MDL for DRO in water is 31 mg/l and 14 mg/kg for soils. The MDL for GRO in water is 8 mg/l and 4 mg/kg for soils. For MRO in water, the MDL is 7 mg/l and for soils 10 mg/kg. Future proposed improvements to this method will involve updated software that will allow automatic blank subtraction, automatic calculation of surrogate recoveries and the automatic incorporation of dry weight factors in the final calculations for soils. In addition, a GRO method with a run time of only 24 min will be used routinely when only GRO analyses have to be performed.  相似文献   

12.
吴刚 《昆虫学报》2005,48(4):531-536
采用药膜法研究了福州建新和闽侯上街菜蚜茧蜂Diaeretiella rapae对杀虫剂抗药性的季节性变化。田间菜蚜茧蜂种群在网室内脱离选择压力后对杀虫剂敏感性显著升高。与敏感的F21子代的LC50相比,2002年6月8日至2004年11月18日期间,建新菜区菜蚜茧蜂对甲胺磷、氟虫腈、阿维菌素、氰戊菊酯和吡虫啉的抗性倍数分别为11.8~31.2,9.3~14.7,9.3~35.7,7.0~16.6,和13.3~30.1倍;2003年6月5日至2004年12月17日期间,上街菜区菜蚜茧蜂对甲胺磷、氟虫腈、阿维菌素、氰戊菊酯和吡虫啉的抗性倍数分别为7.5~34.9,4.0~14.3,9.2~38.6,7.8~18.9和12.1~24.2倍。田间菜蚜茧蜂种群在5月份和6月初抗性水平最高,12月份较低,6月末最低,尤其在6月份期间20天内抗性水平急剧下降。研究结果表明,田间菜蚜茧蜂对6种杀虫剂的抗性水平在脱离选择压力后不稳定,且呈显著的季节性变化。表2参25  相似文献   

13.
Recent studies with Nile tilapia have shown divergent results regarding the possibility of selecting on morphometric measurements to promote indirect genetic gains in fillet yield (FY). The use of indirect selection for fillet traits is important as these traits are only measurable after harvesting. Random regression models are a powerful tool in association studies to identify the best time point to measure and select animals. Random regression models can also be applied in a multiple trait approach to analyze indirect response to selection, which would avoid the need to sacrifice candidate fish. Therefore, the aim of this study was to investigate the genetic relationships between several body measurements, weight and fillet traits throughout the growth period and to evaluate the possibility of indirect selection for fillet traits in Nile tilapia. Data were collected from 2042 fish and was divided into two subsets. The first subset was used to estimate genetic parameters, including the permanent environmental effect for BW and body measurements (8758 records for each body measurement, as each fish was individually weighed and measured a maximum of six times). The second subset (2042 records for each trait) was used to estimate genetic correlations and heritabilities, which enabled the calculation of correlated response efficiencies between body measurements and the fillet traits. Heritability estimates across ages ranged from 0.05 to 0.5 for height, 0.02 to 0.48 for corrected length (CL), 0.05 to 0.68 for width, 0.08 to 0.57 for fillet weight (FW) and 0.12 to 0.42 for FY. All genetic correlation estimates between body measurements and FW were positive and strong (0.64 to 0.98). The estimates of genetic correlation between body measurements and FY were positive (except for CL at some ages), but weak to moderate (−0.08 to 0.68). These estimates resulted in strong and favorable correlated response efficiencies for FW and positive, but moderate for FY. These results indicate the possibility of achieving indirect genetic gains for FW and by selecting for morphometric traits, but low efficiency for FY when compared with direct selection.  相似文献   

14.
Soil and wastewater treatment sludge are commonly brought together in mixtures for a variety of beneficial purposes. The mixtures contain bioacidifying (i.e., sulfur-oxidizing) microorganisms that can easily be activated through providing the appropriate substrate and environmental conditions. In this study, contaminated soil and sludge mixtures were subjected to controlled bio-acidification and the impacts of the process on the partitioning of heavy metals, nitrogen, and phosphorus were examined. Three successive bio-acidification cycles resulted in significant leaching of metals from sludge. The leaching results, expressed as fraction of total mass of metals in the sludge, averaged 67% for Cr, 96% for Ni, 24% for Zn; 16% for Cu; 23% for Cd; and 96% for Pb. Bio-acidification of the sludge also converted 28 to 45% of the organic nitrogen into ammonia and increased the soluble orthophosphates fraction of total phosphorus by approximately 18 to 20%. Bio-acidification also resulted in significant metals leaching from the contaminated soils in the soil/sludge mixtures. Soil/sludge mixtures were prepared using six soil particle sizes (less than 0.075?mm to 2.38?mm) contaminated with 22,500?mg/kg Zn, 14,000?mg/kg Pb, 1500?mg/kg Cr, 9500?mg/kg Cu, 1000?mg/kg Ni, and 1000?mg/kg Cd. The addition of metals to the soil inhibited the sulfur-oxidizing microorganisms, preventing bio-acidification in the mixtures containing 4 to 50?g soil in 130?ml sludge, and considerably slowing bio-acidification in the mixtures containing 1 to 3?g soil. Using a mixture that contained 2-g soil samples, three successive bio-acidification cycles resulted in significant cumulative metals leaching results. The leaching results, expressed as percentage of the mass of metals added to the soil, were in the range of 56 to 98% for Cr, 77 to 95% for Zn, 33 to 66% for Ni, 64 to 82% for Cu, and 10 to 33% for Pb, with the higher results in each range belonging to the larger size soil particles. On the other hand, only Cr was leached in neutralized soil samples. The results confirmed the potential for inhibition of the sulfur-oxidizing microorganisms and bio-acidification in contaminated soil/sludge mixtures, and the significant impacts of bio-acidification on the mobility of metals, nitrogen, and phosphorus. In addition, the results confirmed the potential for using controlled bioacidification for removing heavy metals from contaminated soil using the indigenous sulfur oxidizing microorganisms in sludge.  相似文献   

15.
A total of 1922 first generation crossbred cows born between 2005 and 2012 produced by inseminating purebred Israeli Holstein cows with Norwegian Red semen, and 7487 purebred Israeli Holstein cows of the same age in the same 50 herds were analyzed for production, calving traits, fertility, calving diseases, body condition score, abortion rate and survival under intensive commercial management conditions. Holstein cows were higher than crossbreds for 305-day milk, fat and protein production. Differences were 764, 1244, 1231 for kg milk; 23.4, 37.4, 35.6 for kg fat, and 16.7, 29.8, 29.8 for kg protein; for parities 1 through 3. Differences for fat concentration were not significant; while crossbred cows were higher for protein concentration by 0.06% to 0.08%. Differences for somatic cells counts were not significant. Milk production persistency was higher for Holstein cows by 5, 8.3 and 8% in parities 1 through 3. Crossbred cows were higher for conception status by 3.1, 3.6 and 4.7% in parities 1 through 3. Rates of metritis for Holsteins were higher than the crossbred cows by 7.8, 4.6 and 3.4% in parities 1 to 3. Differences for incidence of abortion, dystocia, ketosis and milk fever were not significant. Holstein cows were lower than crossbred cows for body condition score for all three parities, with differences of 0.2 to 0.4 units. Contrary to comparisons in other countries, herd-life was higher for Holsteins by 79 days. A total of 6321 Holstein cows born between 2007 and 2011 were higher than 765 progeny of crossbred cows backcrossed to Israeli Holsteins of the same ages for milk, fat and protein production. Differences were 279, 537, 542 kg milk; 10.5, 17.7, 17.0 kg fat and 6.2, 12.9, 13.2 kg protein for parities 1 through 3. Differences for fat concentration were not significant, while backcross cows were higher for protein percentage by 0.02% to 0.04%. The differences for somatic cell score, conception rate, and calving diseases other than metritis, were not significant. Holstein cows were lower than backcross cows by 1.5% to 2.5% for conception status in parities 1 to 3 and lower for body condition score for parities 1 and 2, with differences in the range of 0.06 to 0.09 units. Culling rates were higher, and herd-life lower for the crossbred cows. The gains obtained in secondary traits for crossbred cows did not compensate for the major reduction in production.  相似文献   

16.
The purpose of this study was to determine taste difference thresholds for monosodium glutamate (MSG) and sodium chloride (NaCl) in pigtail macaques (Macaca nemestrina) and spider monkeys (Ateles geoffroyi). Using a two-bottle preference test of brief duration, three animals of each species were presented with four different reference concentrations of 50, 100, 200, and 400 mM of a tastant and tested for their ability to discriminate these from lower concentrations of the same tastant. The just noticeable differences (JNDs), expressed as Weber ratios (DeltaI/I), were found to range from 0.1 to 0.5 for MSG and 0.2 to 0.45 for NaCl in the pigtail macaques, with a significant tendency for higher Weber ratios with higher reference concentrations. In the spider monkeys, JNDs ranged from 0.15 to 0.4 for MSG and 0.1 to 0.25 for NaCl, with Weber ratios staying fairly constant across the reference concentrations tested. Thus, the JNDs were found to be generally similar in both species and to be at least as low as those found in humans for MSG and NaCl, as well as those found in spider monkeys for sucrose. The results support the assumption that both pigtail macaques and spider monkeys may use differences in perceived intensity of MSG and NaCl as a criterion for food selection.  相似文献   

17.
Alginate–carbon beads were prepared in order to develop a biocompatible matrix for laccase and glucose oxidase immobilization for application in biofuel cell technology. The enzyme loading capacity was high (91%) in pure alginate beads for glucose oxidase. For laccase, the loading capacity was enhanced from 75% to 83% by introducing carbon. Desorption out of the matrix was controlled by the enzymes’ diffusion and reached a plateau after 40 h for laccase and 70 h for glucose oxidase. Two-thirds of both enzymes was irreversibly retained inside the alginate beads. This proportion increased to 80% for laccase in combined alginate/carbon beads. Half-life of the adsorbed enzyme was enhanced to 74 days for laccase in carbon/alginate beads and 45 days for glucose oxidase in pure alginate as compared to 38 days and 23 days for free enzymes, respectively.  相似文献   

18.
Most people live in urban environments and there is a need to produce abundance indices to assist policy and management of urban greenspaces and gardens. While regional indices are produced, with the exception of birds, studies of the differences between urban and rural areas are rare. We explore these differences for UK butterflies, with the intention to describe changes that are relevant to people living in urban areas, in order to better connect people with nature in support of conservation, provide a measure relevant to human well-being, and assess the biodiversity status of the urban environment.Transects walked under the UK Butterfly Monitoring Scheme are classified as urban or rural, using a classification for urban morphological zones. We use models from the Generalised Abundance Index family to produce urban and rural indices of relative abundance for UK butterfly species. Composite indices are constructed for various subsets of species. For univoltine and bivoltine species, where we are able to fit phenomenological models, we estimate measures of phenology and identify urban/rural differences. Trends in relative abundance over the period 1995–2014 are more negative in urban areas compared to rural areas for 25 out of 28 species. For the composite indices, all trends are negative, and they are significantly more negative for urban areas than for rural areas. Analysis of phenological parameters shows butterflies tend to emerge earlier in urban than in rural areas. In addition, some fly longer in urban than in rural areas, whereas in other cases the opposite is the case, and hypotheses are proposed to account for these features.Investigating new urban/rural indicators has revealed national declines that are stronger for urban areas. For continued monitoring, there is a need for an urban butterfly indicator, and for this to be evaluated and reported annually. We explain how this may be interpreted, and the relevance for other monitoring schemes. The results of this paper, including the phenological findings, shed new light on the potentially deleterious effects of urbanisation and climate change, which require suitable monitoring and reporting to support policy and management, for example of urban greenspaces and gardens.  相似文献   

19.
Data are presented on patterns of failure and delay in making initial treatment contact after first onset of a mental disorder in 15 countries in the World Health Organization (WHO)''s World Mental Health (WMH) Surveys. Representative face-to-face household surveys were conducted among 76,012 respondents aged 18 and older in Belgium, Colombia, France, Germany, Israel, Italy, Japan, Lebanon, Mexico, the Netherlands, New Zealand, Nigeria, People''s Republic of China (Beijing and Shanghai), Spain, and the United States. The WHO Composite International Diagnostic Interview (CIDI) was used to assess lifetime DSM-IV anxiety, mood, and substance use disorders. Ages of onset for individual disorders and ages of first treatment contact for each disorder were used to calculate the extent of failure and delay in initial help seeking. The proportion of lifetime cases making treatment contact in the year of disorder onset ranged from 0.8 to 36.4% for anxiety disorders, from 6.0 to 52.1% for mood disorders, and from 0.9 to 18.6% for substance use disorders. By 50 years, the proportion of lifetime cases making treatment contact ranged from 15.2 to 95.0% for anxiety disorders, from 7.9 to 98.6% for mood disorders, and from 19.8 to 86.1% for substance use disorders. Median delays among cases eventually making contact ranged from 3.0 to 30.0 years for anxiety disorders, from 1.0 to 14.0 years for mood disorders, and from 6.0 to 18.0 years for substance use disorders. Failure and delays in treatment seeking were generally greater in developing countries, older cohorts, men, and cases with earlier ages of onset. These results show that failure and delays in initial help seeking are pervasive problems worldwide. Interventions to ensure prompt initial treatment contacts are needed to reduce the global burdens and hazards of untreated mental disorders.  相似文献   

20.
There is growing international interest in better managing soils to increase soil organic carbon (SOC) content to contribute to climate change mitigation, to enhance resilience to climate change and to underpin food security, through initiatives such as international ‘4p1000’ initiative and the FAO's Global assessment of SOC sequestration potential (GSOCseq) programme. Since SOC content of soils cannot be easily measured, a key barrier to implementing programmes to increase SOC at large scale, is the need for credible and reliable measurement/monitoring, reporting and verification (MRV) platforms, both for national reporting and for emissions trading. Without such platforms, investments could be considered risky. In this paper, we review methods and challenges of measuring SOC change directly in soils, before examining some recent novel developments that show promise for quantifying SOC. We describe how repeat soil surveys are used to estimate changes in SOC over time, and how long‐term experiments and space‐for‐time substitution sites can serve as sources of knowledge and can be used to test models, and as potential benchmark sites in global frameworks to estimate SOC change. We briefly consider models that can be used to simulate and project change in SOC and examine the MRV platforms for SOC change already in use in various countries/regions. In the final section, we bring together the various components described in this review, to describe a new vision for a global framework for MRV of SOC change, to support national and international initiatives seeking to effect change in the way we manage our soils.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号