逆转录-聚合酶链反应检测鼠肝炎病毒方法的建立

建立特异、灵敏的逆转录聚合酶链反应（RT－PCR）技术,结合碱性磷酸酶标记的探针杂交检测鼠肝炎病毒（MHV）,采用MHV－3,MHV－A59病毒株感染DBT细胞,37℃培养,待细胞出现病变时收集提取病毒RNA。依据MHV基因序列设计一对高度保守区特异性引物,进行RT－PCR扩增,结果可见147bp的鼠肝炎病毒产物特异扩增带。敏感性实验检测到10pg的鼠肝炎病毒RNA,同时用ELISA方法对照。结果提示应用RT—PCR技术结合探针杂交检测鼠肝炎病毒。具有简便、快速、灵敏等优势。本研究在国内尚未见报道。     

小鼠肝炎病毒RT-PCR检测方法的建立和比较研究

用小鼠肝炎病毒的MHV1、MHV3、A59、JHM四株分别感染DBT细胞 ,收获病毒 ,提取病毒RNA。依据小鼠肝炎病毒的病毒基因、M结构蛋白、mRNA的基因保守区分别设计出多对引物 ,按各对引物的条件建立了RT -PCR方法 ,比较了不同引物敏感性和特异性 ,并比较选择与MHV同为冠状病毒的传染性支气管炎 (IBV)标准株和野毒株没有交叉的最佳特异引物。敏感性实验检测到 1 0pg的小鼠肝炎病毒RNA。     

小鼠肝炎病毒N基因的原核表达和免疫活性初步分析

目的对小鼠肝炎病毒（mouse hapetitis virus）A59毒株核蛋白（N）基因进行了克隆、表达和重组蛋白的免疫活性分析。方法根据GenBank公布的MHV-A59 N基因序列（AY700211）,设计了一对特异性引物,通过RT-PCR扩增出N基因的主要抗原片段NS,将目的片段纯化后与pGEM-T-easy载体连接得到重组质粒pTN,双酶切回收目的基因片段克隆到原核表达载体pGEX-6p-1中,构建了重组质粒pGEX-NS,转化大肠杆菌BL21,用IPTG进行了诱导表达。对表达裂解物进行SDS-PAGE和Western-blotting验证。结果表达产物相对分子质量约56×103与预期相符;能与MHV阳性血清发生特异性反应,出现单一条带。结论原核表达的NP（S）蛋白有良好的抗原性,为检测小鼠肝炎病毒抗体的ELISA检测方法的研究奠定了基础。     

利用两种不同引物扩增金龟子COⅠ序列片段的研究与对比

利用两类不同的引物,即通用引物(L1490,H2198)与特异引物(Pat,Jerry)分别对4种常见金龟子线粒体细胞色素C氧化酶I(COⅠ)基因片段序列进行扩增和测序,获得长度为689 bp与775 bp的序列。对测序结果进行遗传距离分析,并构建了4种金龟子系统进化树。结果表明,特异引物扩增序列的遗传距离在种内稳定性与种间的差异性都明显优于通用引物扩增序列,利用特异引物扩增序列所构建的系统进化树最符合实际情况,因此利用特异引物扩增序列更能够准确的对金龟子进行分类。     

利用两种不同引物扩增金龟子COI序列片段的研究与对比

利用两类不同的引物,即通用引物(L1490,H219S)与特异引物(Pat,Jerry)分别对4种常见金龟子线粒体细胞色素C氧化酶I(COI)基因片段序列进行扩增和测序,获得长度为689 bp与775 bp的序列.对测序结果进行遗传距离分析,并构建了4种金龟子系统进化树.结果表明,特异引物扩增序列的遗传距离在种内稳定性与种间的差异性都明显优于通用引物扩增序列,利用特异引物扩增序列所构建的系统进化树最符合实际情况,因此利用特异引物扩增序列更能够准确的对金龟子进行分类.     

牛β-酪蛋白5′端上游调控序列的克隆和序列分析

该文用PCR扩增了牛β－酪蛋白基因5′－端上游调控序列,并对其进行了克隆和序列分析。采集成年母牛肝,提取DNA。在牛β－酪蛋白基因外显子1和上游调控区内设计引物,扩增其上游调控序列。两条引物长均为19个核苷酸,引物间跨度为635bp。以牛肝DNA为模板,进行PCR扩增,扩增产物在2％琼脂糖凝胶上电泳,可见特异的目的条带。从凝胶中回收目的片段,克隆到pGEM－T载体中。重组质粒提取DNA,进行序列分析。测序结果与文献发表的类似序列相比,仅有4个碱基不同,同源性达99．4％。表明获得了牛β－酪蛋白基因5′－端上游调控序列的克隆。     

蛇毒类凝血酶Calobin cDNA的设计合成与克隆

人工合成了朝鲜蝮蛇(Agkistrodon cnliginosus)类凝血酶Calobin的编码序列。在设计引物时选择大肠杆菌和酵母茵的偏爱密码子,通过相互搭桥的方式,将全长为789bp的编码序列分成14个片段,每个片段长度为78个碱基左右,采用“核心模板法”并加以改进,通过4次PCR延伸反应,得到了全长基因。经扩增后回收目的片段,双酶切后连接到克隆载体,将得到的转化子酶切鉴定后,任意选择6个克隆进行双向测序,得到了全序列正确的3个克隆。本工作为类凝血酶在大肠杆菌和酵母系统中的高效表达奠定了基础,也为长度为700-900bp片段的合成提供了帮助。     

垃圾填埋场中厌氧真菌18S rDNA的PCR扩增及鉴定

采用机械破壁法直接从来自 7个不同地区的垃圾填埋场滤液样本中提取真菌DNA ,应用真菌通用引物NS1和NS8扩增 18SrDNA(约 180 0bp) ,多聚酶链式反应 (PCR)产物的琼脂糖凝胶电泳结果表明所有的样本均得到了扩增 ;以PCR产物作为模板 ,采用厌氧真菌Chytridiomycetes科的专用引物Chyt 719和Chyt 15 5 3进行二次PCR扩增 (约 85.7bp) ,该阳性扩增产物克隆和测序结果首次表明在食草动物瘤胃中存在的厌氧真菌Chytridiomycetes也存在于垃圾填埋场中 ,且为Neocallimastix属     

黑发菌核糖体DNA小亚基片段的序列测定

用Rusketal 于 1995年设计的引物SMNUR10 1和Whiteetal 1990年设计的引物NS4对黑发菌 (Co matrichanigra)的rDNA小亚基片段进行了PCR扩增 ,并将扩增产物克隆到大肠杆菌JM10 9中进行测序 ,测得的序列长度为 115 4bp。同时将其与多头绒泡菌的相应序列进行了比较 ,其序列同源性为 6 6 %。     

Analysis of retinal and 3-dehydroretinal in the retina by high-pressure liquid chromatography

A sensitive analytical method was developed in order to study the rhodopsin-porphyropsin system in the eye. Oximes of 11-cis-retinal, all-trans-retinal, 11-cis-3-dehydroretinal, and all-trans-3-dehydroretinal were determined quantitatively by high-pressure liquid chromatography. This method was applied to the analysis of retinal and 3-dehydroretinal in the retinas of bullfrog and goldfish. The results agreed with those obtained from the bleaching kinetics of visual pigment extracted with detergent. A reliable result is obtained if the tissue contains more than 5 pmol of retinal (or 3-dehydroretinal). The chromophore composition could be determined in the eye of a small freshwater prawn, Palaemon pancidence, using 50 pmol of 11-cis-retinal and no 3-dehydroretinal.     

Enthalpy array analysis of enzymatic and binding reactions

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. The combination of the small size of the detectors and the ability to perform measurements in parallel results in a significant reduction of sample volume and measurement time compared with conventional calorimetry. We have made significant improvements in the technology by reducing the temperature noise of the detectors and improving the fabrication materials and methods. In combination with an automated measurement system, the advances in device performance and data analysis have allowed us to develop basic enzyme assays for substrate specificity and inhibitor activity. We have also performed a full titration of 18-crown-6 with barium chloride. These results point to future applications for enthalpy array technology, including fragment-based screening, secondary assays, and thermodynamic characterization of leads in drug discovery.     

Neurotoxic and haemolytic activity of a protein isolated from Bacillus thuringiensis var. israelensis crystals

Abstract Two proteins (protein A and B) were isolated from Bacillus thuringiensis var. israelensis crystals using ion-exchange chromatography. Protein A was found to have haemolytic and neurotoxic activities. Inhibition of nerve conduction in the sixth abdominal ganglion of the American cockroach was observed following application of protein A. Protein A was found to be haemolytic to human and rabbit erythrocytes. Protein B did not exhibit any of these activities.     

In vivo models of angiogenesis

The process of building new blood vessels (angiogenesis) and controlling the propagation of blood vessels (anti-angiogenesis) are fundamental to human health, as they play key roles in wound healing and tissue growth. More than 500 million people may stand to benefit from anti- or pro-angiogenic treatments in the coming decades [National Cancer Institute (USA), Cancer Bulletin, volume 3, no. 9, 2006]. The use of animal models to assay angiogenesis is crucial to the search for therapeutic agents that inhibit angiogenesis in the clinical setting. Examples of persons that would benefit from these therapies are cancer patients, as cancer growth and spread is angiogenesis-dependent, and patients with aberrant angiogenesis in the eye, which may lead to blindness or defective sight. Recently, anti-angiogenesis therapies have been introduced successfully in the clinic, representing a turning point in tumor therapy and the treatment of macular degeneration and heralding a new era for the treatment of several commonly occurring angiogenesis-related diseases. On the other hand, pro-angiogenic therapies that promote compensatory angiogenesis in hypoxic tissues, such as those subjected to ischemia in myocardial or cerebral hypoxia due to occluding lesions in the coronary or cerebral arteries, respectively, and in cases of poor wound healing, are also being developed. In this review, the current major and newly introduced preclinical angiogenesis assays are described and discussed in terms of their specific advantages and disadvantages from the biological, technical, economical and ethical perspectives. These assays include the corneal micropocket, chick chorioallantoic membrane, rodent mesentery, subcutaneous (s.c.) sponge/matrix/alginate microbead, s.c. Matrigel plug, s.c. disc, and s.c. directed in vivo angiogenesis assays, as well as, the zebrafish system and several additional assays. A note on quantitative techniques for assessing angiogenesis in patients is also included. The currently utilized preclinical assays are not equivalent in terms of efficacy or relevance to human disease. Some of these assays have significance for screening, while others are used primarily in studies of dosage-effects, molecular structure activities, and the combined effects of two or more agents on angiogenesis. When invited to write this review, I was asked to describe in some detail the rodent mesenteric-window angiogenesis assay, which has not received extensive coverage in previous reviews.     

Ecological tannin assays: a critique

Summary The concept of protein precipitation potential has recently been introduced by Wisdom et al. (1987) as a means to combine chemical and protein precipitation assays of tannins for ecological studies. The definition of protein precipitation potential was not theoretically rigorous, and data analysis was obscure. Our attempts to repeat the tannin extraction procedure gave incomplete recovery (24% loss of quebracho) of condensed tannins, the only type considered by Wisdom et al. In contrast we found their method efficient for hydrolysable tannins (80% recovery of tannic acid) which were undetected by their chemical assay of tannins. Their protein precipitation assay was confounded by chemical interference from both types of tannins. We conclude with recommendations for this type of analysis.     

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.     

MEASUREMENTS OF PHYTOPLANKTON-PROTEIN CONTENT WITH THE HEATED BIURET-FOL1N ASSAY1,2

The heated biuret-Folin method for determining protein consistently measures 90% of the total nitrogen of filtered algae samples as protein-N without the need of mechanical disruption as long as the heating period in biuret is 100 min at 100 C. Data indicate this protein assay measures total protein on all species tried and for naturally occurring mixtures of species plus detritus. Dilute algal suspensions with as little as 0.05 μg-atom particulate protein N.liter ^-1 can he concentrated by fltration on glass fiber filters to 1.0 μg-atom particulate protein-N per filter, the optimal amount of sample for a 5 ml volume of biuret. The filtered algae samples can be stored for several weeks frozen before assaying, if necessary.     

Measurement of enzyme kinetics and inhibitor constants using enthalpy arrays

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. Compared with conventional calorimetry, enthalpy arrays achieve a significant reduction of sample volume and measurement time through the combination of the small size of the detectors and ability to perform measurements in parallel. The current capabilities of the technology for studying enzyme-catalyzed reactions are demonstrated by determining the kinetic parameters for reactions with three model enzymes. In addition, the technology has been used with two classes of enzymes to determine accurate inhibitor constants for competitive inhibitors from measurements at a single inhibitor concentration.