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Uhrf1基因重组腺病毒载体构建及其在小鼠心肌细胞DNA损伤修复中的作用研究
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作者 江南 王驰寅 +1 位作者 聂宇 王珏 《中国病理生理杂志》 CAS CSCD 北大核心 2024年第2期238-243,共6页
目的:构建携带小鼠泛素样同源域和环指结构域1(Uhrf1)基因的重组腺病毒载体,验证Uhrf1基因在原代乳鼠心肌细胞中的表达情况,并探究其在过氧化氢(H_(2)O2)诱导的心肌细胞DNA损伤中的作用。方法:利用PCR扩增小鼠Uhrf1基因的编码序列,将其... 目的:构建携带小鼠泛素样同源域和环指结构域1(Uhrf1)基因的重组腺病毒载体,验证Uhrf1基因在原代乳鼠心肌细胞中的表达情况,并探究其在过氧化氢(H_(2)O2)诱导的心肌细胞DNA损伤中的作用。方法:利用PCR扩增小鼠Uhrf1基因的编码序列,将其酶切后插入pADM-CMV-C-FH载体,获得重组腺病毒质粒ADM-Uhrf1。将该质粒转染至HEK293T细胞包装成重组腺病毒颗粒,数代扩增后进行腺病毒的纯化及滴度检测。分离25只1日龄ICR小鼠原代心肌细胞,分为两组,以感染复数(MOI)为50的比例分别感染ADM-Uhrf1及ADM-control(ADMCtrl),通过Western blot及免疫荧光染色验证重组腺病毒介导的UHRF1蛋白的表达,并利用H_(2)O2诱导心肌细胞DNA损伤,进而探究Uhrf1在DNA损伤修复过程中的作用。结果:通过壳蛋白免疫法检测得到的ADM-Uhrf1病毒滴度为1.8×10^(13) pfu/L。Western blot验证显示UHRF1蛋白表达水平显著升高(P<0.05),免疫荧光染色显示UHRF1主要表达在细胞核内,且Uhrf1的过表达能够显著抑制DNA损伤标志物磷酸化组蛋白H_(2)A变异体(γH_(2)AX)蛋白的表达(P<0.01)。结论:成功构建了携带小鼠Uhrf1基因的重组过表达腺病毒载体,并通过腺病毒递送系统在心肌细胞中实现了Uhrf1的过表达,且Uhrf1的过表达有效减轻了H_(2)O2诱导的心肌细胞DNA损伤。 展开更多
关键词 Uhrf1基因 腺病毒载体 质粒 心肌细胞 dna损伤
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Plasmid DNA Analysis of Pathogenic Escherichia coli in Musk Deer 被引量:11
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作者 罗燕 程建国 +3 位作者 郑士华 赵翠 李蓓 李敏 《Agricultural Science & Technology》 CAS 2009年第3期22-25,共4页
[Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 p... [Objective] The pathogenic Escherichia coli in musk deer was classified at molecular level to provide basic materials for molecular epidemiology of pathogenic Escherichia coli in musk deer. [Method] Plasmids from 24 pathogenic Escherichia coli in musk deer were extracted by the Lysis Triton method, and then identified by single enzyme digestion with three endonucleases of Hind Ⅲ, EcoR Ⅰ and BamH Ⅰ. [Result] The yield rate of plasmids was 91.6%, and 24 pathogenic Escherichia coli in musk deer had the identical or similar plasmid profiles. [Conclusion] Plasmid DNA analysis offers scientific basis for molecular epidemiology of pathogenic Escherichia coli in musk deer in Sichuan Institute of Musk Deer Breeding. 展开更多
关键词 Musk deer Pathogenic Escherichina coil plasmid dna plasmid profile
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指数补料联合升温发酵法提高治疗性HPV DNA疫苗产量的研究进展
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作者 李晓丽 张晓朋 +4 位作者 刘磊 李明珠 赵超 张秀军 魏丽惠 《中国医药导刊》 2024年第8期748-754,共7页
宫颈癌是全球女性第四大常见癌症。目前上市的HPV疫苗均为预防性疫苗,对已感染HPV人群无治疗效果。HPV感染率仍然很高。近年来,科学家日益关注治疗性HPV疫苗的研发,可诱导细胞免疫杀死已感染HPV的细胞。治疗性HPV DNA疫苗以其低成本、... 宫颈癌是全球女性第四大常见癌症。目前上市的HPV疫苗均为预防性疫苗,对已感染HPV人群无治疗效果。HPV感染率仍然很高。近年来,科学家日益关注治疗性HPV疫苗的研发,可诱导细胞免疫杀死已感染HPV的细胞。治疗性HPV DNA疫苗以其低成本、稳定性好的特点备受青睐。为降低治疗性HPV DNA疫苗的成本,目前多采用大肠杆菌高密度发酵生产。本研究使用具有热敏感特性的DNA质粒特性的治疗性HPV DNA疫苗,采用摇瓶培养载有DNA质粒的大肠杆菌,30℃培养一定时间后,分别升温至37℃或42℃,结果显示,升温至42℃组比升温至37℃组的质粒产量明显升高;采用发酵方式培养大肠杆菌,30℃培养一定时间后,升温至42℃进一步提高质粒的拷贝数;分别探究恒速补料和指数补料两种方式联合升温发酵策略对DNA质粒产量的影响,结果显示,相较于恒速补料,指数补料可显著提高升温发酵过程中DNA质粒的产量,从125.76 mg·L^(-1)提升至619.94 mg·L^(-1)。发酵得到的治疗性HPV DNA疫苗可在293T细胞中高效表达,并能在体内引起HPV抗原特异性细胞免疫反应。本研究认为指数补料联合升温发酵策略对其他DNA质粒的发酵也有一定的借鉴意义,也为CAR-T等细胞产品、RNA相关产品提供高质量DNA质粒提供参考。 展开更多
关键词 人乳头瘤病毒(HPV) 治疗性HPV dna疫苗 热敏型质粒 升温发酵 恒速补料 指数补料
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质粒DNA Fast NGS测序方法的开发和应用
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作者 宋辉 曹文刚 +1 位作者 肖晓文 杜军 《生物技术进展》 2024年第4期594-600,共7页
质粒DNA是最常用的基因运载工具,在基因合成技术中扮演着至关重要的角色。如何实现合成质粒DNA的准确且快速检测,是确保基因组完整性和提高基因合成效率的关键。尽管基于一代DNA的测序方法,其准确性已成为行业标准,但在检测通量、检测... 质粒DNA是最常用的基因运载工具,在基因合成技术中扮演着至关重要的角色。如何实现合成质粒DNA的准确且快速检测,是确保基因组完整性和提高基因合成效率的关键。尽管基于一代DNA的测序方法,其准确性已成为行业标准,但在检测通量、检测速度和检测成本等方面仍然存在局限性,这促使科学家们不断寻求新的解决方案。基于生物酶库,开发了DNA建库酶TN5,建立了高通量质粒DNA检测方案——Fast NGS。利用不同长度、不同质量的质粒DNA样本评估了Fast NGS的可行性,并对质粒DNA样本进行了高通量测序,最后对比了Fast NGS与Sanger测序的效率。结果表明,DNA建库酶TN5蛋白的纯度和质量符合二代测序要求。Fast NGS适用于3~8 kb基因合成质粒的测序检测,其检测通量高达2500个·12 h-1,测序成功率超过95%,测序准确性与一代测序相当,并且无明显序列偏好性。Fast NGS实现了质粒DNA的高通量、快速且低成本检测,为基因合成技术的发展提供了新的方向。 展开更多
关键词 Fast NGS 质粒dna 高通量测序 TN5
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In Vitro Antioxidant and Radio Protective Activities of Lycopene from Tomato Extract against Radiation—Induced DNA Aberration
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作者 Safaiatul Islam Abu Hena Mostofa Kamal +2 位作者 Md. Ziaur Rahman Protul Kumar Roy A.Y.K. Md. Masud Rana 《Journal of Biosciences and Medicines》 2024年第2期202-213,共12页
Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurri... Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurring antioxidants that come from plants, as synthetic antioxidants are toxic, carcinogenic and problematic for the environment. Lycopene is one of the carotenoids, a pigment that dissolves in fat and has antioxidant properties. Materials and Methods: The antioxidant and free radical scavenging activity were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The impact of lycopene on bacteria (E. coli) susceptibility to γ-radiation was examined by radio sensitivity assay. The study also examined the induction of strand breaks in plasmid pUC19 DNA and how lycopene extract protected the DNA from γ-radiation in vitro. Results: At varying concentrations, lycopene demonstrated its ability to scavenge free radicals such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH). IC<sub>50</sub> for lycopene was determined at 112 μg/mL which was almost partial to IC<sub>50</sub> of standard antioxidant L-ascorbic acid. The D<sub>10</sub> value 180 Gy of E. coli was found to be >2-fold higher in the extract-containing lycopene sample than in the extract-free controls. The lycopene extracts inhibited the radiation-induced deterioration of the plasmid pUC19 DNA. At an IC<sub>50</sub> concentration, lycopene provided the highest level of protection. Conclusion: Lycopene functions as an efficient free radical scavenger and possible natural antioxidant source. For cancer patients and others who frequently expose themselves to radiation, lycopene may be a useful plant-based pharmaceutical product for treating a variety of diseases caused by free radicals. 展开更多
关键词 Radio Protective ANTIOXIDANTS Free Radical dna Damage pUC19 plasmid Gamma Irradiation DPPH
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质粒DNA实验室规模化制备工艺 被引量:1
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作者 贲培玲 陈容前 +1 位作者 孙淼 施向阳 《山东第一医科大学(山东省医学科学院)学报》 CAS 2023年第3期202-208,共7页
目的 探索实验室规模生产质粒DNA(plasmid DNA,pDNA)的制备工艺。方法 选用质粒pEGFP-N1E. coli Stbl3菌株,用最陡爬坡试验(plackett-burman,PB)筛选出影响质粒产量最显著因素,响应面法优化重组菌高产发酵条件。采用碱裂解,浓缩质粒,通... 目的 探索实验室规模生产质粒DNA(plasmid DNA,pDNA)的制备工艺。方法 选用质粒pEGFP-N1E. coli Stbl3菌株,用最陡爬坡试验(plackett-burman,PB)筛选出影响质粒产量最显著因素,响应面法优化重组菌高产发酵条件。采用碱裂解,浓缩质粒,通过凝胶、亲和、离子等层析分离纯化pDNA,并对所纯化的pDNA进行质量评价。结果 用PB试验和响应面试验设计筛选出的关键因素是:酵母提取物20 g/L,甘油6 g/L,接种物浓度吸光度(optical density,OD)=0.014。在最佳条件下进行3次平行发酵,生物量OD 600达28.07±2.01,质粒产量(21.34±1.31)mg/L。pDNA纯度(A260 nm/A280 nm)为1.91±0.02。内毒素含量小于0.005 EU/g DNA;几乎检测不到蛋白质及细菌基因组DNA残留,达到相关质量标准。结论 本研究采用的制备工艺可生产出高质量的p DNA。 展开更多
关键词 质粒dna 响应面 实验室规模发酵 纯化 质量评价
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混合模式层析分离纯化超螺旋质粒DNA
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作者 张鹏程 谭远志 +3 位作者 孙艳娜 张其磊 姚善泾 林东强 《高校化学工程学报》 EI CAS CSCD 北大核心 2023年第5期806-812,共7页
针对细胞裂解液中的超螺旋质粒DNA(sc pDNA)的分离,以质粒pVAX1为典型对象、采用Capto PlasmidSelect作为混合模式层析介质,探讨了料液中主要成分sc pDNA、开环质粒DNA(oc pDNA)和RNA的吸附行为,优化了分离条件,实现了从成分较为复杂的... 针对细胞裂解液中的超螺旋质粒DNA(sc pDNA)的分离,以质粒pVAX1为典型对象、采用Capto PlasmidSelect作为混合模式层析介质,探讨了料液中主要成分sc pDNA、开环质粒DNA(oc pDNA)和RNA的吸附行为,优化了分离条件,实现了从成分较为复杂的料液中高效分离sc pDNA。考察了上述3种组分的静态吸附,发现在(NH_(4))_(2)SO_(4)浓度c(NH_(4))_(2)SO4为1.9~2.5 mol·L^(-1)时,sc pDNA均具有较高的吸附量,确定c(NH_(4))_(2)SO_(4)=2.5 mol·L^(-1)的料液可直接上样,此时sc pDNA饱和吸附量为每克介质吸附3.3 mg。动态吸附实验发现,sc pDNA穿透略晚于oc pDNA,sc pDNA动态载量为每毫升介质负载2.00 mg,RNA吸附能力明显强于pDNA。进一步优化了洗脱、冲洗和上样量等分离条件,采用c(NH_(4))_(2)SO_(4)=2.5 mol·L^(-1)上样、c(NH_(4))_(2)SO_(4)=1.9 mol·L^(-1)冲洗、(c(NH_(4))_(2)SO_(4)=1.7 mol·L^(-1))+(cNaCl=0.3 mol·L^(-1))洗脱,sc pDNA纯度可达83.9%、同质性高达95.8%、收率为80.6%。结果表明,混合模式层析对sc pDNA选择性好、处理量较大,具有良好的应用价值。 展开更多
关键词 超螺旋质粒dna 吸附 混合模式层析 核酸分离
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Catalytic hydrolysis of phosphate diester (BNPP) and plasmid DNA by mononuclear macrocyclic polyamine metal complexes 被引量:3
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作者 Qing Xiang Xiang Li Qun Zhang +1 位作者 Xiao Qi Yu Ru Gang Xie 《Chinese Chemical Letters》 SCIE CAS CSCD 2009年第5期523-526,共4页
The activities of the catalytic hydrolysis of phosphate diester (BNPP) [bis(p-nitrophenyl)phosphate diester] and plasmid DNA (pUC 18) by mononuclear macrocyclic polyamine metal complexes have been investigated i... The activities of the catalytic hydrolysis of phosphate diester (BNPP) [bis(p-nitrophenyl)phosphate diester] and plasmid DNA (pUC 18) by mononuclear macrocyclic polyamine metal complexes have been investigated in this paper. The results showed that the highest activity in hydrolysis of BNPP was obtained with le--Zn(II) complex (composed of lipophilic group) as catalyst. The hydrolysis rate enhancement is up to 3.64 × 10^4 fold. These metal complexes could effectively promote the cleavage of plasmid DNA (pUC18) at physiological conditions. 展开更多
关键词 Catalytic hydrolysis Macrocyclic polyamine Zn(II) complex BNPP plasmid dna
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Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy 被引量:2
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作者 徐志南 沈文和 +1 位作者 陈灏 岑沛霖 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE EI CAS CSCD 2005年第5期396-400,共5页
Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The ef... Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid pro- ductivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system. 展开更多
关键词 plasmid dna Growth medium Gene therapy
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Extraction of plasmid-like DNA and high-quality total DNA from Porphyra yezoensis 被引量:1
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作者 Guo Baotai1 Dai Jixun1 +3 位作者 Shen Songdong1 Bi Yuping2 Shan Lei2 Li Guangcun2 (1. College of Marine Life Sciences, Ocean University of Qingdao, Qingdao 266003, China 2. Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2000年第2期83-88,共6页
Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA ... Somatic cells were prepared from sea snail enzyme digests of Porphyra yezoensis thalli. Us ing SDS - Proteinase K as extraction solution, total DNA was isolated from the somatic cells. The crude extracts of total DNA were purified with glassmilk, and the resulting DNA was of sufficient quality for digestion of restriction endonuclease. DNA bands were clearly observed in the restriction patterns of EcoRI, PstI and HaeIII respectively. The presence of DNA hands in the restriction pattern of total DNA indicated that the genome of Porphyra yezoensis may be small. Unexpectedly, using guanidinium isoth iocyanate and sarcosyl as extraction solution, a plasmid-like DNA band (2.3 Kb) was directly found in the isolated total DNA of Porphyra yezoensis. A very simple and convenient method for plasmid-like DNA isolation has been established. 展开更多
关键词 Porphyra yezoensis somatic cells total dna glassmilk restriction digestion plasmid like dna
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Optimization on cationic liposome-mediated cell transfection of plasmid DNA 被引量:1
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作者 Mingang Ying Changhua Zhuo Weidong Zang 《The Chinese-German Journal of Clinical Oncology》 CAS 2011年第5期290-292,共3页
Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by ... Objective:The development of gene carriers for efficient gene delivery into cells has attracted growing attention in recent years.The aim of this study was to achieve a better outcome of AAV-293 cells transfection by plasmid DNA.Methods:We studied the optimal condition for higher efficiency of cationic lipid-mediated cell transfection.Four experimental groups were set.Plasmid DNA and liposome were mixed in each groups at different ratios(μg:μL),1:2.5,1:3.5,1:4.0 and 1:5.0,respectively.LacZ gene functioned as reporter gene,measuring the transfection efficiency of the four groups using the method of X-gal staining.Results:When the ratio was 1:3.5,the cell transfection rate was the highest.While the ratio of 1:2.5 recommended by product manual achieve the lowest transfection rate.Their difference had statistical significance.Conclusion:In order to obtain a higher transfection efficiency,optimization on conditions of the ratio of plasmid DNA to liposome is necessary in cell transfection. 展开更多
关键词 cell transfection cationic lipid plasmid dna cell culture transfection efficiency
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Amplification of plasmid DNA bound on soil colloidal particles and clay minerals by the polymerase chain reaction
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作者 CAI Peng HUANG Qiao-yun +3 位作者 LU Yan-du CHEN Wen-li JIANG Dai-hua LIANG Wei 《Journal of Environmental Sciences》 SCIE EI CAS CSCD 2007年第11期1326-1329,共4页
Polymerase chain reaction (PCR) was used to amplify a 600-base pair (bp) sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil (Alfisol) and Red soil (Ultisol), and three different ... Polymerase chain reaction (PCR) was used to amplify a 600-base pair (bp) sequence of plasmid pGEX-2T DNA bound on soil colloidal particles from Brown soil (Alfisol) and Red soil (Ultisol), and three different minerals (goethite, kaolinite, montmorillonite). DNA bound on soil colloids, kaolinite, and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted, 10- and 20-fold. The montmorillonite-DNA complex required at least 100-fold dilution before amplification could be detected. DNA bound on goethite was amplified irrespective of whether the complex was used directly, or diluted 10- and 20-fold. The amplification of mineral-bound plasmid DNA by PCR is, therefore, markedly influenced by the type and concentration of minerals used. This information is of fundamental importance to soil molecular microbial ecology with particular reference to monitoring the fate of genetically engineered microorganisms and their recombinant DNA in soil environments. 展开更多
关键词 ADSORPTION AMPLIFICATION MINERAL PCR plasmid dna soil colloid
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Preparation of Ag/AgBr/TiO_2 as Catalyst Carriers and Its Damage to Plasmid DNA and Tetrahymena
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作者 刘力维 张银龙 《Journal of Wuhan University of Technology(Materials Science)》 SCIE EI CAS 2015年第5期1068-1073,共6页
The composites based on the Ti O2 are potentially used in wetland pollution control. In this work, the biological effect of the Ag/Ag Br/Ti O2/Active carbon(AC) composites was studied on the plasmid DNA and Tetrahymen... The composites based on the Ti O2 are potentially used in wetland pollution control. In this work, the biological effect of the Ag/Ag Br/Ti O2/Active carbon(AC) composites was studied on the plasmid DNA and Tetrahymena membrane. The atomic force micrograph(AFM) images showed that, in the presence of the composites under illumination, most p UC18 DNA molecules showed quite different topography and were opened and relaxed circle shapes. After DNA was catalyzed for 40 min, all supercoiled and circular DNA were changed into the linear DNA molecules. The gel electrophoresis experiment confirmed the results and demonstrated the dynamic process of DNA degradation. ATR-FTIR spectra revealed that amide groups and PO2-of the phospho-lipid phospho-diester on Tetrahymena surface were oxidized in the presence of the composites under illumination. An increase in the fluorescence polarization of DPH was observed, reflecting a significant decrease in membrane fluidity of Tetrahymena. 展开更多
关键词 Ti O2 water treatment Tetrahymena plasmid dna WETLAND
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Delivery of Plasmid DNA into Tumors by Intravenous Injection of PEGylated Cationic Lipoplexes into Tumor-Bearing Mice
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作者 Yoshiyuki Hattori 《Pharmacology & Pharmacy》 2016年第7期272-282,共11页
For systemic injection of cationic liposome/plasmid DNA (pDNA) complexes (cationic lipoplexes), polyethylene glycol (PEG)-modification (PEGylation) of lipoplexes can enhance their systemic stability. In this study, we... For systemic injection of cationic liposome/plasmid DNA (pDNA) complexes (cationic lipoplexes), polyethylene glycol (PEG)-modification (PEGylation) of lipoplexes can enhance their systemic stability. In this study, we examined whether intravenous injection of PEGylated cationic lipoplexes into tumor-bearing mice could deliver pDNA into tumor tissues and induce transgene expression. PEGylation of cationic liposomes could prevent their agglutination with erythrocytes. However, when PEGylated cationic lipoplexes were injected intravenously into tumor-bearing mice, they accumulated in tumor vascular vessels and did not exhibit transgene expression in tumors with both poor and well-developed vascularization. Furthermore, PEGylated cationic lipoplexes of CpG- free pDNA could not increase transgene expression in tumors after intravenous injection. These results suggested that PEGylation could not extravasate cationic lipoplexes from vascular vessels in tumors and abolished transgene expression although it enhanced the systemic stability of cationic lipoplexes by avoiding interactions with blood components such as erythrocytes. Successful delivery of pDNA to tumors by PEGylated cationic liposomes will require a rational strategy and the design of liposomal delivery systems to overcome the issue associated with the use of PEG. 展开更多
关键词 Cationic Liposome LIPOPLEX plasmid dna PEGYLATION TUMOR
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Analysis of heavy-ion-induced DNA strand breaks in plasmid pUC18
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作者 GUO Hui-jun1,LIU Lu-xiang1,LI Jia-cai2,ZHAO Kui3,SUI Li3,ZHAO Lin-shu1,ZHAO Shi-rong1(1.The National Key Facility for Crop Gene Resources and Genetic Improvement,institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China 2.Institute of High Energy Physics,Chinese Academy of Sciences,Beijing 100093,China 3.Department of Nuclear Physics,China Institute of Atomic Energy,Beijing 102413,China) 《湖南农业大学学报(自然科学版)》 CAS CSCD 北大核心 2007年第S1期242-,共1页
Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with po... Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with positive linear-dose-effects;most of sequence changes induced by CR were point mutant.Lithium-ion-beam could induce strand breaks also,but it was only at dose of 20Gy. 展开更多
关键词 dna Analysis of heavy-ion-induced dna strand breaks in plasmid pUC18 CR
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Destruction of <i>Escherichia coli</i>and Broad-Host-Range Plasmid DNA in Treated Wastewater by Dissolved Ozone Disinfection under Laboratory and Field Conditions
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作者 Kyle L. Asfahl Mary C. Savin 《Advances in Microbiology》 2012年第1期1-7,共7页
Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been id... Broad-host-range plasmids are frequently associated with antibiotic resistance genes and can quickly spread antibiotic resistant phenotypes among diverse bacterial populations. Wastewater treatment plants have been identified as reservoirs for broad-host-range plasmids carrying resistance genes. The threat of broad-host-range plasmids released into the environment from wastewater treatment plants has identified the need for disinfection protocols to target broad-host- range plasmid destruction. Here we evaluate the efficacy of dissolved ozone at 2 and 8 mg·L–1 as a primary means for the destruction of broad-host-range plasmid and chromosomal DNA in simulated effluent. Pilot-scale tests using an experimental unit were carried out in municipal wastewater treatment plant effluent and compared with ultraviolet (UV)-irradiation and chlorination methodologies. Genes specific to Escherichia coli (uidA) and IncP broad-host-range plasmids (trfA) were monitored using real-time quantitative polymerase chain reaction (qPCR), and total DNA was monitored using absorbance spectroscopy. In wastewater treatment plant experiments, E. coli qPCR results were compared to a recognized culture-based method (Colilert?) for E. coli. In laboratory experiments, dissolved ozone at 8 mg·L–1 significantly destroyed 93% total, 98% E. coli, and 99% of broad-host-range plasmid DNA. Ozonation, UV-irradiation, and chlorination significantly reduced DNA concentrations and culturable E. coli in wastewater treat- ment plant effluent. Chlorination and UV disinfection resulted in 3-log decreases in culture-based E. coli concentrations in wastewater treatment plant effluent while changes were not significant when measured with qPCR. Only ozonation significantly decreased the IncP broad-host-range plasmid trfA gene, although concentrations of 2.2 × 105 copies trfA·L–1 remained in effluent. Disinfection processes utilizing high dissolved ozone concentrations for the destruction of emerging contaminants such as broad-host-range plasmid and total DNA may have utility as methods to ensure downstream environmental health and safe water reuse become more important. 展开更多
关键词 DISINFECTION Wastewater Treatment Ozone qPCR plasmid Broad-Host-Range plasmid dna ESCHERICHIA COLI
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实时定量PCR测定治疗性HBV DNA疫苗工程菌的质粒拷贝数
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作者 陈孟璋 何星垚 林汉森 《广东药科大学学报》 CAS 2023年第5期87-92,共6页
目的 比较不同的定量方法和总DNA制备方法对实时定量PCR(real-time quantitative PCR, qPCR)测定治疗性HBV DNA疫苗工程菌的质粒拷贝数(plasmid copy number, PCN)的影响,为建立快速简便的治疗性HBV DNA疫苗工程菌的PCN测定方法提供参... 目的 比较不同的定量方法和总DNA制备方法对实时定量PCR(real-time quantitative PCR, qPCR)测定治疗性HBV DNA疫苗工程菌的质粒拷贝数(plasmid copy number, PCN)的影响,为建立快速简便的治疗性HBV DNA疫苗工程菌的PCN测定方法提供参考。方法 通过试剂盒提取法、Triton X-100煮沸法和培养基煮沸法分别制备总DNA样品用于qPCR检测,使用绝对定量方法和相对定量方法计算不同制备方法样品的PCN,并对测定结果进行统计学分析。结果 试剂盒提取法制备的样品经绝对定量和相对定量计算PCN,结果分别为43.10±4.02和42.92±3.98,显著低于Triton X-100煮沸法的结果(69.32±6.51和68.58±6.42)以及培养基煮沸法的结果(75.73±7.46和76.15±7.50)。任一样品制备方法,比较其绝对定量和相对定量结果,差异均无统计学意义。结论 qPCR的绝对定量和相对定量计算方法都适用于治疗性HBV DNA疫苗工程菌PCN测定,Triton X-100煮沸法更适合于总DNA模板制备。 展开更多
关键词 HBV疫苗 dna疫苗 实时定量PCR 质粒拷贝数
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Construction of DNA Vaccine for FMDV P1 Gene and Immunization Experiment
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作者 史秋梅 高桂生 +2 位作者 张艳英 高光平 张东林 《Agricultural Science & Technology》 CAS 2013年第8期1069-1071,共3页
[Objective] This study aimed to construct DNA vaccine of foot-and-mouth disease (FMD).[Method] Plasmid carriers plESZP1 and pUTK3CP1 with PRV were constructed for FMDV P1 gene expression.Mice were immunized,and thei... [Objective] This study aimed to construct DNA vaccine of foot-and-mouth disease (FMD).[Method] Plasmid carriers plESZP1 and pUTK3CP1 with PRV were constructed for FMDV P1 gene expression.Mice were immunized,and their antibody level was detected.The two eukaryotic expression plasmids constructed were transfected into Vero cells.PCR,IFA and Westem-blot were carried out to detect the transcription and expression of the objective gene.Balb/C mice were intramuscularly inoculated with the DNA plasmid which expressed the target gene correctly,and the antibody level in mice was detected by the means of ELISA and serum neutralization (SN).[Result] DNA plasmid carrying P1 gene which encodes FMDV capsid protein caused specific body fluid immunoreaction in mice,and the antibody level of anti-FMDV had no difference in the mice induced by the two recombinant plasmids.[Conclusion] This study lays a foundation for evaluating the genetically modified vaccine by immunizing animals with recombinant PRV containing the FMDV P1 gene and recombinant virus. 展开更多
关键词 FMDV P1 gene dna plasmid Immunization experiment
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一种新型可用于DNA分子量标准的质粒构建 被引量:9
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作者 韦柳静 韦宇拓 +2 位作者 黄小凤 韦旭钦 黄日波 《生物技术》 CAS CSCD 2004年第5期33-35,共3页
将首尾带有EcoRI酶切位点的 2 .0kb、1.5kb、1.0kb、0 .75kb、0 .5kb、0 .2 5kb六个长度的片段逐步连接到pGEM - 3zf(+)质粒上的BamHI位点中 ,构建的质粒用EcoRI进行单酶切 ,经电泳可以获得七条DNA带与设计结果完全一致 ,可用于DNA电泳... 将首尾带有EcoRI酶切位点的 2 .0kb、1.5kb、1.0kb、0 .75kb、0 .5kb、0 .2 5kb六个长度的片段逐步连接到pGEM - 3zf(+)质粒上的BamHI位点中 ,构建的质粒用EcoRI进行单酶切 ,经电泳可以获得七条DNA带与设计结果完全一致 ,可用于DNA电泳试验中分子量标准。 展开更多
关键词 新型 dna 分子量标准 质粒
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碱裂解法提取质粒DNA的研究 被引量:16
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作者 杨献光 齐志广 +1 位作者 赵宝存 马闻师 《生物技术通报》 CAS CSCD 2003年第6期24-26,共3页
对质粒DNA及其结构、形态和功能进行了综述 ,介绍了碱裂解法提取质粒DNA的基本步骤 ,并对其提取过程中的注意事项进行了剖析 。
关键词 质粒dna 提取 碱裂解法 结构 形态 功能
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