共查询到19条相似文献，搜索用时 109 毫秒
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《科学中国人》2015,(26)
目的构建SLO原核表达质粒,重组蛋白的诱导表达并纯化。方法提取链霉菌溶血素O模板DNA,PCR法扩增slo基因。构建融合表达重组质粒p GEX-6p-1-slo和pet32a-tev-slo,将正确的重组质粒转化至大肠杆菌BL21和大肠杆菌BL21-DE3,使用异丙基硫代βD半乳糖苷(IPTG)诱导表达重组融合蛋白。采用亲和层析纯化重组蛋白,切除标签后,再通过亲和层析纯化,获取SLO重组蛋白。结果 PCR扩增出slo基因,基因片段(1700 bp)与理论一致。经SDSPAGE,蛋白质印迹显示重组蛋白相对分子质量为55 k Da,与数据库中的相对分子质量结果相符。结论成功构建了slo原核表达质粒,表达并纯化了SLO重组蛋白。 相似文献
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目的:建立原位石英晶体微天平(QCM)-环介导恒温扩增(LAMP)快速检测大肠杆菌O157特异基因的方法。方法:在环介导恒温核酸扩增(LAMP)技术平台引入石英晶体微天平(QCM)技术,采用巯基化试剂分子组装方法,将LAMP反应体系中的4个引物之一固定于QCM电极上,在安装所述电极的QCM检测池中配置O157 LAMP反应体系进行环介导恒温核酸扩增,用QCM仪器在线检测频率变化,判断LAMP反应是否发生,进而判断体系中是否存在O157特异基因。结论:该方法检测O157特异基因特异性强、灵敏度高,并且操作简便,有望发展成为快速检测O157特异基因的有效手段。 相似文献
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食品微生物检验新的、快速简便方法不断地被开发出来,对于各个方法间的一致性暂缺乏统一的评价,本文以酶联免疫方法为备选方法、国家食品卫生标准方法为参考方法,对沙门氏菌和肠出血性大肠埃希氏菌O157进行了检验验证,目的是为不同检验方法之间地的一致性评价提供一定的参考。结果表明,对于沙门氏菌,两种方法检验结果基本一致;而对于肠出血性大肠埃希氏菌O157,两种方法之间有较大的差异,分析表明国家食品卫生标准方法GB／T4789．6不太适合肠出血性大肠埃希氏菌O157的检验。 相似文献
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His-猪生长激素融合基因在大肠杆菌中的表达与纯化 总被引：1，自引：1，他引：1
将通过PCR技术改建并去除了信号肽的猪生长激素cDNA克隆入大肠杆菌表达载体pET-28a( ),构建出能利用Ni-NTAAgarose柱一步纯化表达产物的重组质粒pPGH020。探讨了大肠杆菌中重组猪生长激素的最佳表达条件和纯化方法,制备并纯化了兔抗pGH抗体。SDS鄄PAGE和Westernblot的分析结果表明,26.5kD处有包括His·Taq、thrombin位点序列和pGH序列的融合蛋白特异条带。凝胶扫描估测猪生长激素的表达量占大肠杆菌胞浆蛋白的17.3%左右。 相似文献
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采用含有 K88、K99、987p 抗原决定簇的三株大肠杆菌作为全菌抗原对乳牛进行免疫 ,取免疫牛初乳 ,经盐析、透析获得免疫球蛋白 G( Ig G)。体外抑菌实验结果表明免疫牛初乳中的特异性 Ig G较普通 Ig G具有显著的抑菌作用 相似文献
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目的:研究检验科对微生物检验质量相关的影响因素以及病原菌的耐药性。方法:本研究回顾分析了2017年1月到2018年1月期间我院检验科收检的657份标本,通过回顾调查微生物检验报告的方法从人员、标本、操作等方面调查分析检验质量的相关影响因素。根据CLSI标准通过常规方式对病原菌进行分离并使用k-B纸片扩散法对病原菌进行药敏实验。结果:在各类型标本中痰液的检测准确率最高,为92.31%(P0.05);对各影响因素分析发现,操作人员的影响最大,占42.42%(P0.05);大肠杆菌、肺炎克雷伯菌对喹诺酮类药物以及头孢菌素的耐药率相对较高,耐药率均大于50%;金黄色葡萄球菌对喹诺酮类药物、红霉素、四环素、青霉素的耐药性均大于50%。结论:检验人员及标本质量均是影响检验准确率的影响因素,应规范检验人员的操作技能、提高标本在各环节的质量,同时根据各病原菌的耐药情况选择药物,提高临床疗效。 相似文献
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采用FTA滤膜从蜜饯中直接提取模板DNA,根据金黄色葡萄球菌的nuc基因、沙门氏菌的phoP基因、福氏志贺氏菌的ipaH基因,设计3对特异性引物进行多重PCR,并对反应条件进行优化。建立的三重PCR具有准确、快速、高效的特点,为同时检测蜜饯中金黄色葡萄球菌、沙门氏菌、福氏志贺氏菌提供了新的方法。 相似文献
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鸡蛋是重要的日常必需食品,其储存品质和质量安全备受重视。本研究从新鲜收集的鸡蛋表面分离到多种细菌,经细菌培养、菌落纯化、基因组DNA提取、PCR扩增、电泳检测、16S rDNA序列测定、BLAST序列比对等步骤,鉴定出3株新型Staphylococcus agnetis菌。该类细菌属于一类较新的葡萄球菌,革兰氏染色呈阳性,可能与肉鸡、蛋鸡炎症相关,并可能影响鸡蛋储存期限和食品质量安全性。本研究为进一步开展相关的食品微生物污染及质量控制奠定了新的基础。 相似文献
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Anubha Paliwal Nivedita Bir Sowmya Raghuraman T. L. P. Reddy P. Usha Sarma 《Indian journal of clinical biochemistry : IJCB》1999,14(2):129-134
Oligonucleotide primers were synthesised based on the gene sequence of an 18 kDa allergen/antigen ofA. fumigatus isolated from a pathogenic strain. Polymerase chain reaction (PCR) was carried out using the forward and reverse primers
and genomic DNA ofA. fumigatus, A. flavus andA. niger as template. This resulted in a PCR product of 480 bp with onlyA. fumigatus. The absence of PCR product inA. flavus andA. niger with the primers of Asp fl facilitated use of these primers for detection ofA. fumigatus in clinical specimens of patients. The results were compared with microscopy, culture and serology. Application of PCR test
to clinical samples of aspergillosis patients is discussed. 相似文献
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Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis
BackgroundLawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate.ResultsBatch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers.ConclusionsConsidering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.How to cite: Salazar S, Gutiérrez N, Sánchez O, et al. Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis. Electron J Biotechnol 2021.https://doi.org/10.1016/j.ejbt.2021.01.002 相似文献
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Sheshadri Narayanan 《Indian journal of clinical biochemistry : IJCB》1999,14(1):40-48
The apo E gene located on chromosome 19 in humans is polymorphic. The three apo E isoforms E2, E3, and E4 are coded by three
common alleles of the gene. The amyloid plaques in brains of Alzheimer disease (AD) patients are known to contain apo E. There
is an increased prevalence of E4 allele in AD patients. apo E exhibits increased binding to a peptide Aβ deriving from amylold
precursor protein. apo E, the risk factor for late AD disease is unable to prevent formation of paired helical filaments which
in turn destabilizes neuronal microtubules.
A variety of molecular techniques are available for apo E genotyping using DNA amplified by the polymerase chain reaction
(PCR). The high guanine to cytosine content of apo E is problematic to the extent that the yield of PCR product and hybridization
stringency can be compromised. The specificity of diagnosis of late-onset AD can be improved when results of apo E genotyping
are evaluated together with clinical criteria. 相似文献
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Two simple gold nanoparticle (GNP)-based DNA analysis methods using a microfluidic device are presented. In the first method, probe DNA molecules are immobilized on the surface of a self-assembled submonolayer of GNPs. The hybridization efficiency of the target oligonulceotides was improved due to nanoscale spacing between probe molecules. In the second method, target DNA molecules, oligonulceotides or polymerase chain reaction (PCR) amplicons, are first bound to GNPs and then hybridized to the immobilized probe DNA on a glass slide. With the aid of GNPs, we have successfully discriminated, at room temperature, between two PCR amplicons (derived from closely related fungal pathogens, Botrytis cinerea and Botrytis squamosa) with one base-pair difference. DNA analysis on the microfluidic chip avoids the use of large sample volumes, and only a small amount of oligonucelotides (8 fmol) or PCR products (3 ng), was needed in the experiment. The whole procedure was accomplished at room temperature in 1 h, and apparatus for high temperature stringency was not required. 相似文献
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Christopher R. Phaneuf Nikita Pak D. Curtis Saunders Gregory L. Holst Joav Birjiniuk Nikita Nagpal Stephen Culpepper Emily Popler Andi L. Shane Robert Jerris Craig R. Forest 《Biomicrofluidics》2015,9(4)
Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. 相似文献