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Establishment and Application of a Real-time PCR Method for Detecting stx2 Gene in Shiga Toxin-producing Escherichia coli(STEC)
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作者 汪伟 张雪寒 +6 位作者 王润 何孔旺 温立斌 倪艳秀 周俊明 王小敏 李彬 《Agricultural Science & Technology》 CAS 2014年第9期1473-1477,共5页
[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli (STEC). [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR ... [Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli (STEC). [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples. 展开更多
关键词 Shiga toxin-producing E. colr Shiga toxin 2 gene Real-time PCR
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Gene therapy for human colorectal carcinoma using human CEA promoter controlled bacterial ADP-ribosylating toxin genes:PEA and DTA gene transfer 被引量:18
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作者 CAO Guang Wen 1, QI Zhong Tian 1, PAN Xin 1, ZHANG Xiao Qin 1, MIAO Xiao Hui 1, FENG Yan 1, LU Xin Hua 1, Shigeki Kuriyama 2 and DU Ping 1 《World Journal of Gastroenterology》 SCIE CAS CSCD 1998年第5期25-28,共4页
AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aerugi... AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa. PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA) promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out.RESULTS Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma. 展开更多
关键词 colorectal neoplasms gene therapy gene transfer carcinoembryonic antigen pseudomonas EXOtoxin A DIPHTHERIA toxin A
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Isolation and identification of a marine killer yeast strain YF07b and cloning of the gene encoding killer toxin from the yeast 被引量:1
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作者 WANG Xianghong CHI Zhenming LI Jing 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2007年第6期101-108,共8页
It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculcttus. The marine yeast strain ... It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculcttus. The marine yeast strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The gene encoding killer toxin in the marine yeast strain YF07b was amplified by PCR technology. After sequencing, the results show that an open reading frame, consisting of 1 281 bp, encoded a presumed protein of 427 amino acids. The sequence of the cloned gene was found to have 99% match with that of the gene encoding killer toxin in Pichia anomalas strain K. A signal peptide including 17 amino acids appeared in the N-terminal domain of the killer toxin. Therefore, the mature protein consisted of 410 amino acids, its molecular mass was estimated to be 47.4 ku and its isoelctronic point was 4.5. 展开更多
关键词 pathogenic yeast CRAB marine killer yeast identification killer toxin gene cloning
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Antimicrobial Effects of Plant Compounds against Virulent <i>Escherichia coli</i>O157:H7 Strains Containing Shiga Toxin Genes in Laboratory Media and on Romaine Lettuce and Spinach
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作者 Javier R. Reyna-Granados Lynn A. Joens +2 位作者 Bibiana Law Mendel Friedman Sadhana Ravishankar 《Food and Nutrition Sciences》 2021年第4期392-405,共14页
<span style="font-family:Verdana;"><i><span style="font-family:Verdana;"><i></span></i></span><span style="font-family:Verdana;"><span s... <span style="font-family:Verdana;"><i><span style="font-family:Verdana;"><i></span></i></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">Escherichia coli</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> strains produce Shiga-toxins Stx-1 and Stx-2 that contribute to their virulence. The objective was to evaluate antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, cinnamaldehyde, citral) and plant-extracts (green tea polyphenols, apple skin, black tea, decaffeinated black tea, grapeseed and pomace extracts) against </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">E. coli</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> O157:H7 strains containing </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">Stx</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">-</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">1</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> and </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">Stx</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">-</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">2</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> genes, as determined by Multiplex Polymerase Chain Reaction, </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">in vitro</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> and on leafy greens. Antimicrobials at various concentrations in sterile PBS were added to bacterial cultures (</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">~</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">3</span></span></span><span><span><span style="font-family:""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">-</span></span></span><span><span><span style="font-family:""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">4 logs CFU/ml), mixed thoroughly, and incubated at 37</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">&deg;</span></span></span><span><span><span style="font-family:""><span style="font-family:Verdana;">C</span><span style="font-family:Verdana;">. Surviving bacteria were enumerated at 0, 1, 3, 5 and 24 h. The most effective essential oil (oregano oil;0.5%) and plant extract (green tea;3%) were evaluated against </span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">E. coli</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> O157:H7 on romaine lettuce and spinach stored at 4</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">&deg;</span></span></span><span><span><span style="font-family:""><span style="font-family:Verdana;">C</span><span style="font-family:Verdana;"> for 7 days. Microbial survival was a function of the concentration of antimicrobials and incubation times. All antimicrobials reduced bacterial population to below detection levels </span></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">in vitro</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">;however, essential oils and active components exhibited greater activity than plant extracts. Oregano oil and green tea reduced </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">E. coli</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> O157:H7 on lettuce and spinach to below detection. Plant-based antimicrobials have the potential to protect foods against </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i></span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;">E. coli</span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><i><span style="font-family:Verdana;"></i></span></i></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;"> O157:</span></span></span><span><span><span style="font-family:""> </span></span></span><span style=" 展开更多
关键词 E. coli O157:H7 Shiga toxin genes Romaine Lettuce SPINACH Inactivation Essential Oils Plant Extracts
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Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin
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作者 Dilzamar V.Nascimento Odir A.Dellagostin +5 位作者 Denise C.S.Matos Douglas McIntosh Raphael Hirata Jr. Geraldo M.B.Pereira Ana Luíza Mattos-Guaraldi Geraldo R.G.Armoa 《American Journal of Molecular Biology》 2017年第4期176-189,共14页
Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is u... Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay. 展开更多
关键词 Recombinant BCG Diphtheria toxin dtb gene Park Williams 8(PW8) Corynebacterium diphtheriae rDTBPW8 pUS977 Vector
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食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立 被引量:4
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作者 曹冬梅 袁慕云 +1 位作者 许龙岩 曹际娟 《食品安全质量检测学报》 CAS 2013年第5期1451-1457,共7页
目的建立对副溶血性弧菌(Vibrio parahaemolyticus)特异性检测toxR(跨膜转录激活蛋白)基因和tdh(热稳定性直接溶血素)毒力基因的Taqman探针双色荧光PCR检测方法。方法根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman... 目的建立对副溶血性弧菌(Vibrio parahaemolyticus)特异性检测toxR(跨膜转录激活蛋白)基因和tdh(热稳定性直接溶血素)毒力基因的Taqman探针双色荧光PCR检测方法。方法根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102cfu/mL。结论该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。 展开更多
关键词 副溶血性弧菌 toxR基因 tdh基因 双色荧光PCR 检测
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副溶血性弧菌致病性标志基因tdh的实时荧光定量聚合酶链式反应的建立 被引量:2
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作者 王宏萍 周晓明 +3 位作者 张继伦 姜婷 李雯雯 鲍依稀 《第四军医大学学报》 北大核心 2008年第24期2232-2235,共4页
目的:建立一个用于副溶血性弧菌致病性定量测定的检测体系.方法:以多组tdh基因的保守序列为基础,设计实时荧光定量PCR扩增适用的引物和TaqMan探针.以定量方式提取副溶血性弧菌基因组DNA,以tdh+菌株为阳性对照,建立实时荧光定量PCR方法.... 目的:建立一个用于副溶血性弧菌致病性定量测定的检测体系.方法:以多组tdh基因的保守序列为基础,设计实时荧光定量PCR扩增适用的引物和TaqMan探针.以定量方式提取副溶血性弧菌基因组DNA,以tdh+菌株为阳性对照,建立实时荧光定量PCR方法.以tdh荧光定量结果与绝对菌数浓度作参比,以确定tdh基因在菌群中的拷贝水平.结果:使用正向序列5′-CGAAG ATGTT TATGG TCAAT C-3′(位置在467~487bp处),反向序列5′-ACCGC TGCCA TTG-TA TAGTC T-3′(位置在571~551bp处),TaqMan探针5′-FAM-TGACA TCCTA CATGA CTGTG AAC-ECLIPSE-3′(位置在517~539bp处)建立一个实时荧光定量PCR反应,目的片段长度105bp.建立反应的DNA拷贝数对数值与Ct值线性关系为y=-3.144logx+43.229(r=0.997,P<0.001).建立了菌液A值与显微镜下绝对菌数浓度的相关关系为y=2.0452x+6.2845(r=0.7828,P<0.01).根据tdh基因拷贝数与绝对菌数相比可计算tdh基因的单菌体拷贝量.结论:建立了一个定量副溶血性弧菌tdh基因拷贝水平的实时荧光定量PCR检测方法,可应用于副溶血性弧菌致病性的标准化定量测定体系. 展开更多
关键词 弧菌 副溶血性 聚合酶链反应 tdh基因
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副溶血性弧菌tdh基因的分子信标PCR技术检测 被引量:2
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作者 万成松 谭翰清 温文川 《中国公共卫生》 CAS CSCD 北大核心 2006年第12期1475-1477,共3页
目的采用分子信标PCR技术进行副溶血性弧菌tdh基因检测。方法在反应体系中加入分子信标探针。对13株副溶血性弧菌和其他细菌分别进行tdh基因实时和终点法荧光检测。结果2株副溶血性弧菌和阳性质粒的终点法检测荧光值分别为114.9,95.2... 目的采用分子信标PCR技术进行副溶血性弧菌tdh基因检测。方法在反应体系中加入分子信标探针。对13株副溶血性弧菌和其他细菌分别进行tdh基因实时和终点法荧光检测。结果2株副溶血性弧菌和阳性质粒的终点法检测荧光值分别为114.9,95.2,90.0。实时PCR检测的CT值分别为26.2,26.8,32.0。其他肠道细菌终点法检测荧光值为47.0~69.1;CT值〉32.0或无值,与琼脂糖电泳分析结果一致。结论分子信标PCR技术可以准确、快速、实时、简便地进行副溶血性弧菌tdh基因检测。 展开更多
关键词 副溶血性弧菌(VP) 分子信标 tdh基因 实时PCR
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荧光定量PCR法检测副溶血弧菌tdh基因的表达差异 被引量:4
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作者 王淑娜 方维焕 《畜牧与兽医》 北大核心 2008年第9期9-13,共5页
以pvuA为内标基因,运用实时荧光定量PCR检测不同来源以及不同应激条件下副溶血弧菌热稳定直接溶血素基因tdh的表达量。pvuA和tdh基因的荧光定量PCR融解曲线分析表明,两者均为特异性扩增。尽管相同来源的不同菌株间tdh表达量存在显著差异... 以pvuA为内标基因,运用实时荧光定量PCR检测不同来源以及不同应激条件下副溶血弧菌热稳定直接溶血素基因tdh的表达量。pvuA和tdh基因的荧光定量PCR融解曲线分析表明,两者均为特异性扩增。尽管相同来源的不同菌株间tdh表达量存在显著差异,副溶血弧菌临床分离株的tdh mRNA平均表达量显著高于海产品分离株((57.2比13.8)。在pH4.0、0.5%和8%NaCl应激条件下,临床株ZJ2和海产品分离株FJ14A的tdh mRNA表达量显著高于对照组;另一海产品分离株KP34在8%NaCl条件下的表达量显著提高,而低pH应激时tdh mRNA的表达量显著降低。结果表明,不同副溶血弧菌分离株的tdh mRNA表达差异显著,临床分离株的tdh mRNA表达量总体上高于海产品分离株,副溶血弧菌在不同应激条件下主要表现为tdh mRNA表达上调。 展开更多
关键词 副溶血弧菌 荧光定量PCR tdh基因 表达差异
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聚合酶链反应检测粪便中的副溶血性弧菌的TDH基因
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作者 任少堂 刘军民 +2 位作者 秦一中 汪伟业 俞守义 《上海医学检验杂志》 1997年第4期193-195,共3页
副溶血性弧菌是细菌性腹泻的主要致病因子.本研究建立一种快速标本处理和聚合酶链反应(PCR)诊断方法,扩增副溶血性弧菌的热稳定直接溶血素(TDH)基因。对一组56例腹泻患者粪便标本进行了检测,经细菌培养证实为副溶血性弧菌的9份... 副溶血性弧菌是细菌性腹泻的主要致病因子.本研究建立一种快速标本处理和聚合酶链反应(PCR)诊断方法,扩增副溶血性弧菌的热稳定直接溶血素(TDH)基因。对一组56例腹泻患者粪便标本进行了检测,经细菌培养证实为副溶血性弧菌的9份标本,PCR法均阳性;培养法明性的39例中,有7例检测到TDH基因.统计学表明两种方法检测结果相当一致,但检出能力PCR法显著高于培养法。本法简便、快速、特异,适宜临床应用. 展开更多
关键词 副溶血性弧菌 聚合酶链反应 tdh基因
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Helicobacter pylori virulence genes 被引量:30
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作者 Anja Sterbenc Erika Jarc +1 位作者 Mario Poljak Matjaz Homan 《World Journal of Gastroenterology》 SCIE CAS 2019年第33期4870-4884,共15页
Helicobacter pylori(H.pylori)is one of the most important human pathogens,infecting approximately half of the global population.Despite its high prevalence,only a subset of H.pylori infected individuals develop seriou... Helicobacter pylori(H.pylori)is one of the most important human pathogens,infecting approximately half of the global population.Despite its high prevalence,only a subset of H.pylori infected individuals develop serious gastroduodenal pathology.The pathogenesis of H.pylori infection and disease outcome is thus thought to be mediated by an intricate interplay between host,environmental and bacterial virulence factors.H.pylori has adapted to the harsh milieu of the human stomach through possession of various virulence genes that enable survival of the bacteria in the acidic environment,movement towards the gastric epithelium,and attachment to gastric epithelial cells.These virulence factors enable successful colonization of the gastric mucosa and sustain persistent H.pylori infection,causing chronic inflammation and tissue damage,which may eventually lead to the development of peptic ulcers and gastric cancer.Numerous studies have focused on the prevalence and role of putative H.pylori virulence genes in disease pathogenesis.While several virulence factors with various functions have been identified,disease associations appear to be less evident,especially among different study populations.This review presents key findings on the most important H.pylori virulence genes,including several bacterial adhesins and toxins,in children and adults,and focuses on their prevalence,clinical significance and potential relationships. 展开更多
关键词 Helicobacter pylori Virulence genes Disease association CHILDREN ADULTS Outer membrane proteins Bacterial toxins
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Effects of p75 neurotrophin receptor knockout on axonal regeneration in a mouse model of facial nerve injury 被引量:3
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作者 Fenghe Zhang Ping Huang +1 位作者 Pishan Yang Xue Zhang 《Neural Regeneration Research》 SCIE CAS CSCD 2009年第8期565-569,共5页
BACKGROUND: Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remai... BACKGROUND: Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE: To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING: A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS: Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS: In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES: Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS: Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice (P 〈 0.05). The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice (P 〈 0.01). CONCLUSION: p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury. 展开更多
关键词 p75 neurotrophin receptors cholera toxin B subunit fast blue REgeneRATION MOUSE gene knockout
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Rice Blast Resistance of Transgenic Rice Plants with Pi-d2 Gene 被引量:2
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作者 CHEN De-xi CHEN Xue-wei +3 位作者 LEI Cai-lin MA Bing-tian WANG Yu-ping LI Shi-gui 《Rice science》 SCIE 2010年第3期179-184,共6页
Resistance to rice blast of transgenic rice lines harboring rice blast resistance gene Pi-d2 transformed from three different expression vectors of pCB6.3kb, pCB5.3kb and pZH01-2.72kb were analyzed. Nine advanced-gene... Resistance to rice blast of transgenic rice lines harboring rice blast resistance gene Pi-d2 transformed from three different expression vectors of pCB6.3kb, pCB5.3kb and pZH01-2.72kb were analyzed. Nine advanced-generation transgenic rice lines with Pi-d2 gene displayed various resistance to 39 rice blast strains, and the highest disease-resistant frequency reached 91.7%. Four early-generation homozygous transgenic lines with Pi-d2 gene exhibited resistance to more than 81.5% of 58 rice blast strains, showing the characteristic of wide-spectrum resistance. The transgenic embryonic calli selected by the crude toxin of rice blast fungus showed that the callus induction rate of immature embryo from transgenic rice plants decreased as the concentration of crude toxin in the culture medium increased. When the concentration of crude toxin reached 40%, the callus induction rate of immature embryo from transgenic lines was 49.3%, and that of the receptor control was 5%. The disease incidence of neck blast of the transgenic rice lines in fields under induction was 0% to 50%, indicating that the rice blast resistance of transgenic rice lines is much higher than that of the receptor control. 展开更多
关键词 transgenic rice disease resistance gene rice blast resistant spectrum crude toxin callus induction
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Secretory Expression of Recombinant Diphtheria ToxinMutants in B. Subtilis 被引量:1
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作者 周建华 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 1999年第4期253-256,共4页
Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118... Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku. 展开更多
关键词 B. Subtilis diphtheria toxin Western blot recombinant protein gene expression MUTATION
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Purification and Structural Analysis of a Selective Toxin Fraction Produced by the Plant Pathogen Setosphaeria turcica 被引量:4
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作者 ZHANG Li-hui DONG Jin-gao +1 位作者 WANG Chao-hua LI Zheng-ping 《Agricultural Sciences in China》 CAS CSCD 2007年第4期452-457,共6页
Thirteen fractions from the pathogenic plant fungus Setosphaeria turcica race 1 were separated and collected using high performance liquid chromatography (HPLC). Their toxic activities were assayed through leaf punc... Thirteen fractions from the pathogenic plant fungus Setosphaeria turcica race 1 were separated and collected using high performance liquid chromatography (HPLC). Their toxic activities were assayed through leaf puncturing on corn differentials (OH43, OH43Ht1, OH43Ht2, and OH43HtN), and the results revealed that eight fractions were toxic and fraction 6 was specifically toxic to OH43Ht1, which could be taken as a gene-selective toxin fraction. Fraction 6 was finely purified via HPLC and condensed by freeze desiccation. Its chemical structure was analyzed with EI-MS, IR, HMBC, ^1H-NMR, and two-dimensional NMR. The results suggested that fraction 6 contained an unsaturated double bond, carbonyl and methylene groups with molecular weight of 142. 展开更多
关键词 Setosphaeria turcica gene-specific toxin fraction structure analysis high performance liquid chromatography (HPLC)
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Specific killing effect of diphtheria toxin A fragment under control of DF3 promotor on human breast cancer cells
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作者 Ming Cai Wenguang Huang Wei Luo Sheng Pan Tao Yin 《The Chinese-German Journal of Clinical Oncology》 CAS 2007年第2期200-203,共4页
Objective: To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods: Constructing recombinant expression v... Objective: To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods: Constructing recombinant expression vector PGL3-DF3-DTA and transfecting it into human breast cancer cells of DF3 positive and negative. By means of RT-PCR to measure the expression of DTA in human breast cancer cells. MTT color-imetry was used to examine the effect of PGL3-DF3-DTA on growth of human breast cancer cells. By experiment on nude mice to observe the killing effect of PGL3-DF3-DTA on human breast cancer cells. Results: Recombinant expression vector PGL3-DF3-DTA was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Conclusion: Recombinant expression vector PGL3-DF3-DTA could produce specific killing effect on human breast cancer cell line of DF3 positive. 展开更多
关键词 DF3 diphtheria toxin A fragment gene expression gene therapy breast cancer
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基于多交叉置换扩增和纳米生物传感技术快速检测肺炎支原体方法的建立
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作者 肖飞 郑宝英 +8 位作者 徐文健 伏瑾 黄小兰 孙春荣 贾楠 张裕 许峥 周娟 王毅 《遵义医科大学学报》 2024年第5期513-521,共9页
目的建立一种简单、灵敏、快速的肺炎支原体(MP)检测方法,并对其应用性进行验证和评价。方法利用多交叉置换扩增(MCDA)技术对肺炎支原体特异基因CARDS毒素基因进行扩增,利用侧流免疫层析生物传感(LFB)技术读取扩增结果,命名该方法为MP-M... 目的建立一种简单、灵敏、快速的肺炎支原体(MP)检测方法,并对其应用性进行验证和评价。方法利用多交叉置换扩增(MCDA)技术对肺炎支原体特异基因CARDS毒素基因进行扩增,利用侧流免疫层析生物传感(LFB)技术读取扩增结果,命名该方法为MP-MCDA-LFB。分析扩增反应在60~67℃(间隔1℃)的扩增效率,筛选最适反应温度;分析分别扩增10、20、30、40 min时能够检测到的最低核酸浓度,筛选最佳反应时间。利用10倍系列稀释的肺炎支原体核酸分析MP-MCDA-LFB方法的灵敏度和检测限,利用35株非肺炎支原体菌株分析MP-MCDA-LFB方法的特异性。利用MP-MCDA-LFB方法检测80份疑似MP感染的临床样本,并与RT-PCR法检测结果进行比较,分析MP-MCDA-LFB方法的临床应用性。结果MP-MCDA-LFB能够实现对肺炎支原体CARDS毒素基因的快速检测。其最佳反应温度为63℃,最短反应时间为40 min,整个检测过程可在1 h内。MP-MCDA-LFB方法具有较高的灵敏度和特异性,其检测限低至45 ng/L,与其他临床表现相似的病原体无交叉反应,特异性为100%。MP-MCDA-LFB方法从80份临床样本中检出45份阳性样本(56.3%),检出率与RT-PCR方法一致。结论本研究建立的以CARDS毒素基因为靶标的MP-MCDA-LFB检测方法具有简单、快速、灵敏度高、特异性强的优点,在基层医疗机构和现场检测具有较好的应用潜力。 展开更多
关键词 肺炎支原体 多交叉置换扩增技术 侧流免疫层析生物传感技术 CARDS毒素基因 RT-PCR
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肉鸡源产气荚膜梭菌的分离鉴定及耐药性分析 被引量:2
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作者 黄紫贝 罗健雅 +4 位作者 赵以恒 李蒙 苏思元 赵振华 刘文博 《中国畜牧兽医》 CAS CSCD 北大核心 2024年第3期1298-1307,共10页
【目的】对江苏某大型白羽肉鸡场疑似产气荚膜梭菌感染造成大规模死亡的病例进行确诊。【方法】无菌采集病死鸡肝脏、脾脏、肺脏、肾脏及肠道等病变组织,通过厌氧培养进行细菌分离和纯化,革兰染色镜检对分离菌株进行初步鉴定,通过16S r... 【目的】对江苏某大型白羽肉鸡场疑似产气荚膜梭菌感染造成大规模死亡的病例进行确诊。【方法】无菌采集病死鸡肝脏、脾脏、肺脏、肾脏及肠道等病变组织,通过厌氧培养进行细菌分离和纯化,革兰染色镜检对分离菌株进行初步鉴定,通过16S rRNA基因序列分析确定属和演化关系,通过PCR扩增毒素基因确定分离株毒素型,通过动物致病性试验和药敏试验,确定其致病性和耐药表型并检测耐药基因。【结果】从4只病死鸡不同脏器中分离到了9株疑似菌,分离菌在产气荚膜梭菌鉴定培养基上呈黑色,16S rRNA分析得出9株分离菌与产气荚膜梭菌相似性最高可达99.60%。16S rRNA基因系统进化树显示,4株分离株与巴基斯坦鸡源产气荚膜梭菌(GenBank登录号:MN365136.1)具有较近的亲缘性,5株分离株与南非鸡源产气荚膜梭菌(GenBank登录号:OR494055)具有较近的亲缘性。毒素分型结果显示,5株为A型,2株为F型,2株为E型。致病性试验结果显示,小鼠全部死亡,表明该菌具有致死性。药敏试验结果显示,9株分离株均对头孢类抗生素敏感,耐药基因分析表明大环内酯类erm(B)(77.8%)和四环素类tetA(P)(55.6%)、tetB(P)(66.7%)为主要耐药基因。【结论】本研究从同一只病死鸡中分离到不同毒素型产气荚膜梭菌,且分离菌耐药情况较严重。研究结果为兽医临床鸡产气荚膜梭菌病防治提供了新的思路。 展开更多
关键词 产气荚膜梭菌 毒素基因 耐药性
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市售肉品产志贺毒素大肠杆菌污染情况与耐药性分析——以泰安市为例 被引量:1
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作者 张天宁 李文阳 +4 位作者 董鹏程 毛衍伟 杨啸吟 罗欣 朱立贤 《中国动物传染病学报》 CAS 北大核心 2024年第3期119-127,共9页
产志贺毒素大肠杆菌(STEC)是一类产生一种或一种以上志贺毒素的具有强致病能力的食源性病原菌,现已是威胁人类健康的重要公共卫生问题之一。本文以山东省泰安市为调查点,对商超和农贸市场零售肉中产志贺毒素大肠杆菌(STEC)的污染情况进... 产志贺毒素大肠杆菌(STEC)是一类产生一种或一种以上志贺毒素的具有强致病能力的食源性病原菌,现已是威胁人类健康的重要公共卫生问题之一。本文以山东省泰安市为调查点,对商超和农贸市场零售肉中产志贺毒素大肠杆菌(STEC)的污染情况进行调研,170份零售肉样品中9份样品检出STEC阳性,检出率为5.3%;其中商超和农贸市场的检出率分别为3.1%和6.7%,无显著性差异(P>0.05)。通过聚合酶链式反应(PCR)研究24株STEC分离菌株的血清型、毒力基因的携带情况。24株STEC中12株确定O血清型,分别为O26(10株)和O45(2株)。主要携带的毒力基因为stx1,hlyA(62.5%,15/24)和stx2(37.5%,9/24)基因,eaeA基因未检出。通过纸片扩散法对24株产志贺毒素大肠杆菌菌株进行耐药性检测,分离株对麦迪霉素的耐药率最高91.7%,对阿莫西林、氨苄西林的耐药率均为37.5%,对头孢西丁和多粘菌素敏感。综上所述,泰安市零售畜禽肉品中存在STEC不同程度的污染,其中猪肉、牛肉引起食源性疾病风险较大。本研究为相关部门加强市售肉品中STEC的监控提供一定的指导。 展开更多
关键词 零售肉 STEC 耐药性 血清型 毒力基因
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2株公园湖水源非O1/O139群霍乱弧菌的分离鉴定和致病性分析 被引量:1
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作者 廖佳玮 施霁桢 +4 位作者 李金鑫 任志浩 张越 谭丽琼 苏敬良 《中国兽医杂志》 CAS 北大核心 2024年第7期49-57,共9页
近年来,非O1/O139群霍乱弧菌(NOVC)引起人和动物感染时有报道。本试验于2021年3月—2022年4月连续采集圆明园遗址公园湖水样本进行细菌分离培养,经16S rRNA序列分析和溶血性检测对分离株进行鉴定,并通过细胞毒性试验和小鼠感染试验对分... 近年来,非O1/O139群霍乱弧菌(NOVC)引起人和动物感染时有报道。本试验于2021年3月—2022年4月连续采集圆明园遗址公园湖水样本进行细菌分离培养,经16S rRNA序列分析和溶血性检测对分离株进行鉴定,并通过细胞毒性试验和小鼠感染试验对分离株的致病性进行检测;采用PCR方法对分离株的毒力相关基因ctxA、ctxB、tcpA、hlyA、rtxA、rtxC和chxA进行检测;通过系统发育进化分析对分离株的chxA基因全长进行分析;通过药敏试验对分离株的药物敏感性进行检测。结果显示,共分离鉴定出2株NOVC,NOVC分离株在血琼脂培养基上生长呈现明显的溶血现象;经过滤除菌的细菌培养液上清有溶细胞作用,对体外培养的Vero细胞具有极强的毒性作用,可引起细胞萎缩和脱落;分离株培养物和培养液上清经腹腔注射均可致死小鼠,对小鼠的半数致死量(LD_(50))为10^(7.23)CFU。PCR检测结果显示,分离株hlyA、rtxA、rtxC和chxA基因呈阳性,提示细菌的致病性可能与毒力因子的表达相关。对chxA基因的全长分析结果显示,2株分离株均可编码II型ChxA毒素,其催化域与I型毒素高度同源,但在相应区域缺失1段5个氨基酸片段(^(619)AIAKE^(623))。药敏试验结果显示,2株分离株均对环丙沙星、恩诺沙星和阿米卡星敏感,均对复方新诺明耐药。结果表明,公园水源性NOVC分离株携带多种毒力相关基因,产生溶血素等溶细胞毒素,可致死小鼠,对抗菌药呈多重耐药,对水生动物和环境的公共卫生具有潜在的威胁。 展开更多
关键词 非O1/O139群霍乱弧菌 致病性 毒力基因 ChxA毒素
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