Feline Ubiquitin Fusion Protein Genes     

Kano  R.  Kubota  A.  Nakamura  Y.  Watanabe  S.  Hasegawa  A. 《Veterinary research communications》2001,25(8):615-622

Using cDNA from a CRFK cell line as a template, PCR amplification was performed with the Ub1S and poly(dT) primers to isolate feline ubiquitin genes. Sequencing of the 495 bp PCR fragment revealed that the putative amino acids induced by this fragment gave a fusion protein consisting of a ubiquitin polypeptide (76 amino acids) and an extension protein of ribosomal proteins L40 (52 amino acids). The putative amino acid sequence of ubiquitin was identical to those of humans, rats and pigs.The recombinant glutathione S-transferase (GST)–feline ubiquitin fusion proteins were produced in Escherichia coli and purified. The fusion proteins had a molecular weight of about 42 kDa and were detected by immunoblot assay with rabbit anti-ubiquitin antiserum.The mRNAs from heat-shocked and non-heat-shocked cells were subjected to RT-PCR (Ub1S and poly(dT) primers) analysis. The molecular weights of the ubiquitinated proteins in heat-shocked CFRK cells were between 18 kDa and 24 kDa by immunoblot assay.These results suggested that there were more ubiquinated proteins in the heat-shocked CRFK cells than in the pre-heat-shocked cells.  相似文献   

K步稳定的鞋印花纹图像自动聚类     

王新年  舒莹莹 《上海交通大学学报(农业科学版)》2016,(5):574-587

目的 鞋印是刑事侦查的重要物证之一,如何对积累的大量鞋印花纹图像进行自动归类管理是刑事技术迫切需要解决的问题之一。与其他类图像不同,鞋印花纹图像具有种类多但数目未知、同类花纹分布不均匀且同类花纹数目少的特点。基于鞋印花纹图像的这些特点,用目前典型的聚类算法对鞋印花纹图像集进行聚类,并不能取得很好的效果。在对鞋印花纹图像进行分析的基础上,提出一种K步稳定的鞋印花纹图像自动聚类算法。方法 对已标记的鞋印花纹图像进行统计发现,各类鞋印花纹之间在特征空间上存在互不相交的区域(本文称为隔离带)。算法的核心思想是寻找各类鞋印花纹之间的隔离带,来将各类分开。过程为:以单调递增或递减的方式调整特征空间中判定两点为一类的阈值,得到数据集的多次划分;若在连续K次划分的过程中,某一类的成员不发生变化,则说明这K次调整是在隔离带中进行的,即聚出一类,并从数据集中删除已标记的数据;选择下一个阈值对剩余的数据集进行划分,输出K步不变的类;依此类推,直到剩余数据集为空,聚类完成。结果 在两类公开测试数据集和实际鞋印花纹数据集上进行实验,本文算法的主要性能指标都超过典型算法,其中在包含5792枚实际鞋印花纹数据集上的聚类准确率和F-Measure值分别达到了99.68%和95.99%。结论 针对鞋印花纹图像特点,提出了一种通过寻找各类之间的隔离带进行自动聚类的算法,并在实际应用中取得了很好的效果。且算法性能受参数的变化以及类的形状影响较小。本文算法同样适用于具有类似特点的其他数据集的自动聚类。  相似文献   

红椿SSR-PCR体系建立和多态性引物筛选          下载免费PDF全文

湛欣  鲁好君  赵帅  陈晓阳  邓小梅 《林业科学研究》2016,29(4):565-570

[目的]本研究旨在建立红椿SSR-PCR最佳反应体系,并筛选适于红椿SSR分析的高多态性引物。[方法]通过L16(45)正交试验设计,确立红椿SSR-PCR最佳反应体系;利用优化后的体系对来自楝科植物的135对SSR引物,在6个不同的红椿居群中进行扩增,筛出能有效扩增的引物并进一步筛选出适于红椿的高多态性引物。[结果](1)10μL基于荧光d UTP的SSR-PCR体系中包含:10×buffer 1.0μL,Taq酶(5 U·μL-1)0.1μL,Mg Cl2(25mmol·L-1)0.8μL,d NTP(200 mmol·L-1)0.025μL,荧光d UTP(1 nmol·μL-1)0.01μL,引物(10 mmol·L-1)0.8μL,DNA模板45 ng剩余用dd H2O补足;(2)筛选出29对能有效扩增的引物,复选后获得了12对适于红椿SSR分析的高多态性引物。[结论]建立了SSR-PCR最佳反应体系并筛选出高多态性引物,为红椿的分子标记等遗传学研究提供了基础。  相似文献   

Genotyping of Bovine Viral Diarrhoea Viruses Isolated from Cattle in Northern Italy     

Falcone E  Cordioli P  Sala G  Tarantino M  Tollis M 《Veterinary research communications》2001,25(2):161-167

Following the first official report of a clinically severe outbreak of bovine viral diarrhoea disease occurring in a farm in northern Italy, which had originated from the use of a live vaccine contaminated with a strain of BVD genotype II virus, a retrospective study on the prevalence of BVDV genotypes in Italy became highly relevant. For this purpose, the genotype of 78 BVDV-positive specimens, obtained in 1998–1999 from dairy cattle in an area near to where the outbreak occurred, was characterized by PCR technology. Two sets of primers, spanning the 5 UTR of BVDV genome, were used sequentially in a first round of RT-PCR, performed on viral RNA extracted directly from 15 clinical samples and 63 BVDV-infected cell-culture fluids; a second PCR assay followed to selectively amplify only BVDV genotype II. All the viruses under study were characterized as BVDV genotype I. As well as contributing to a better understanding of the prevalence of BVDV genotypes in the field, the results of the present study illustrate the possibility that novel BVDV strains can emerge in susceptible animals through the use of contaminated immunobiological products for bovine use.  相似文献   

Characterization of an Indian Bluetongue Virus Isolate by RT-PCR and Restriction Enzyme Analysis of the VP-7 Gene Sequence     

Tiwari AK  Kataria RS  Desai G  Butchaiah G  Bandyopadhyay SK 《Veterinary research communications》2000,24(6):401-409

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A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus     

Pfeffer M  Freyburg MV  Kaaden OR  Beer M 《Veterinary research communications》2000,24(7):491-503

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.  相似文献   

Pathogenicity assays restrict the species Xanthomonas campestris into three pathovars and reveal nine races within X. campestris pv. campestris     

E. Fargier  C. Manceau 《Plant pathology》2007,56(5):805-818

The species Xanthomonas campestris (Vauterin) groups bacteria associated with cruciferous plants. In order to clarify and refine the pathovar and race structures within X . campestris , 47 representative strains of six pathovars were characterized for their pathogenicity on a large host range of Brassicaceae, including all original hosts. Three diseases were observed on tested plants: (i) black rot disease on cruciferous plants; it was proposed that all strains causing black rot on at least one cruciferous plant be grouped in the single pathovar X . c . pv. campestris ; (ii) leaf spot disease caused by X . c . pv. raphani on hosts belonging to the Brassicaceae and Solanaceae; the sequenced strain 756C identified as X . c . pv. armoraciae was included in this pathovar and the existence of another leaf spot disease caused by X . c . pv. armoraciae was not supported; and (iii) bacterial blight of garden stocks caused by X . c . pv. incanae . No plants susceptible to X . c . pv. barbareae were found. Strains that did not induce any symptom on cruciferous plants tested, including their original hosts, were removed from the pathovar scheme and were named X . campestris only. Three new races were described in addition to the six races previously described within X . c . pv. campestris . The sequenced strains ATCC 33913 (CFBP 5241) and Xcc 8004 (CFBP 6650) belonged to race 3 and to race 9 (one of the new races described), respectively.  相似文献   

Use of Degenerate Primers in a RT-PCR Assay for the Identification and Analysis of Some Filamentous Viruses, with Special Reference to Clostero- and Vitiviruses of the Grapevine     

Pasquale Saldarelli  Adib Rowhani  Geoffrey Routh  Angelantonio Minafra  Michele Digiaro 《European journal of plant pathology / European Foundation for Plant Pathology》1998,104(9):945-950

RT-PCR with degenerate primers was used for the screening of the genome of some members of the Closterovirus, Vitivirus and Trichovirus genera. Two sets of primers, targeted to conserved sequences of the heat shock protein 70 homologue of closteroviruses or to the RNA dependent RNA polymerase genes of tricho- and vitiviruses, amplified the expected fragments from total RNA extracts or double-stranded RNAs of infected plants. Amplified cDNAs were cloned, sequenced and phylogenetically analyzed. Results support the allocation of grapevine viruses A, B, D and heracleum latent virus (HLV) in the genus Vitivirus, whereas, the detection of a HSP70 homologue in grapevine leafroll-associated viruses agrees with their assignment in the genus Closterovirus. The use of degenerate primers for the identification of grapevine viruses belonging to Vitivirus and Closterovirus genera is envisaged.  相似文献   

绵羊FecB突变检测方法的建立及高繁滩羊核心群的构建     

周世卫  吴庭杰  王倩  蔡蓓  韩赛铮  吴彦虎  王小龙  陈玉林 《畜牧兽医学报》2022,53(9):2920-2929

旨在建立快速、高效、精准检测FecB突变的荧光qPCR技术,为滩羊多羔性能研究、群体改良及肉用多羔滩羊新品系培育提供技术支撑。基于荧光qPCR技术基本原理,设计特异性引物对,开发基于荧光qPCR检测绵羊FecB突变的方法;对85只已知FecB基因型的健康、适繁雌性滩羊血液基因组DNA样品分别采用PCR-Sanger测序法、TaqMan探针法和荧光qPCR法进行FecB基因分型,统计3种方法的准确率;采用开发的荧光qPCR方法对939只健康、适繁滩羊进行FecB基因分型,统计不同FecB基因型经产母羊的产羔率。结果表明,使用TaqMan探针法、PCR-Sanger测序法和开发的荧光qPCR法对已知FecB基因型样品检测的比较中,荧光qPCR法对各基因型鉴定的准确度均达100%,高于前两者。939只滩羊FecB基因分型结果显示,6只滩羊为FecB突变纯合型（BB）、57只为杂合型（B+）、876只为野生型（++）,BB型、B+型和++型滩羊的基因型频率分别为0.64%、6.07%和93.29%,其中B等位基因频率为0.04,+等位基因频率为0.96;监测母羊群体的产羔数发现,FecB突变纯合型滩羊产羔率为166.67%,突变杂合型滩羊产羔率为153.12%,野生型滩羊产羔率为105.78%,纯合突变型和杂合突变型母羊群体的产羔率极显著高于野生型群体（P<0.01）。综上所述,与TaqMan探针法及PCR-Sanger测序法相比,本研究建立的绵羊FecB突变荧光qPCR分型方法具有简便易行、廉价高效的优点,在绵羊的分子选育中具有较高应用价值;同时本研究证实了滩羊FecB突变可显著提高母羊产羔率,为多羔滩羊的分子选育提供了坚实的技术支撑。  相似文献   

普陀樟SRAP-PCR反应体系的建立     

吕昕谣  赵颖  赵国淼  曾燕如  宋绪忠  杨华 《浙江林业科技》2014,(3)

以舟山群岛的普陀樟（Cinnamomum japonicum var.chenii）新鲜叶片为实验材料,采用L16（45）正交试验设计,对影响普陀樟SRAP-PCR反应的Mg2+浓度、dNTPS、Taq DNA聚合酶、引物和模板DNA用量5因素组合进行了筛选。结果表明：适于普陀樟的SRAP-PCR反应体系（20μL）为2.5 mmol/L Mg2+、0.2 mmol/L dNTPs、1.5 U Taq DNA聚合酶、0.4μmol/L引物、10 ng模板DNA、2μL 10×PCR buffer,该体系位点清晰,扩增稳定;利用该反应体系从100对SRAP引物组合中筛选出多态性好的引物24对。  相似文献