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At present there is a lack of standard criteria for the identification and evaluation of activity of antiviral compounds. Interferon was used to explore comparatively several laboratory methods. Interferon was produced in chick embryos and in chorioallantoic membranes suspended in vitro. Evaluation of interferon activity was performed by several methods: (i) percentage of inhibition of plaque-forming units; (ii) hemagglutinin reduction of challenge virus; (iii) titer of cytopathic effect of challenge virus; and (iv) plaque-inhibition test. The suggested methods for measurement are those which express the titer of challenge virus in plaque-forming units or in hemagglutinating units. 相似文献
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以伴刀豆球蛋白A(ConA)诱导的犬外周血淋巴细胞中提取的总RNA为模板,通过RT PCR的方法克隆扩增出犬α干扰素基因,将所扩增基因克隆于原核表达载体pBV220 并进行测序,结果显示该基因与GenBank上所公布的犬α干扰素基因同源性为100%。将重组表达载体转入宿主菌进行温敏诱导表达,表达产物经SDS PAGE分析,证明目的蛋白以包涵体的形式存在,大小约为19kDa。将表达产物变性、复性、透析、纯化处理后加入犬肾细胞上,用水泡性口炎病毒攻毒,测出重组的CaIFN α具有较高的抗病毒作用,生物活性达到5.11×106 ∪/ ,重组蛋白质的含量约为13 /L。 相似文献
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犬α干扰素基因的高效表达及其活性测定 总被引:1,自引:0,他引:1
以伴刀豆球蛋白A(ConA)诱导的犬外周血淋巴细胞中提取的总RNA为模板,通过RT-PCR的方法克隆扩增出犬α干扰素基因,将所扩增基因克隆于原核表达载体pBV220并进行测序,结果显示该基因与GenBank上所公布的犬α干扰素基因同源性为100%.将重组表达载体转入宿主菌进行温敏诱导表达,表达产物经SDS-PAGE分析,证明目的蛋白以包涵体的形式存在,大小约为19kDa.将表达产物变性、复性、透析、纯化处理后加入犬肾细胞上,用水泡性口炎病毒攻毒,测出重组的CaIFN-α具有较高的抗病毒作用,生物活性达到5.11×10 6∪/㎎,重组蛋白质的含量约为13㎎/L. 相似文献
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干扰素(interferon,IFN)是宿主先天免疫系统的重要组成部分,它是抵抗病原体的第一道防御。为了利用家蚕杆状病毒表达系统表达鸡κ干扰素,根据已报道的序列对鸡κ干扰素的基因序列进行家蚕密码子优化,并利用共转染技术成功构建了表达用重组杆状病毒。将重组杆状病毒感染家蚕,成功获得了大量鸡κ干扰素的表达产物,并通过Western blot技术对产物进行了检测鉴定。用细胞病变抑制法对干扰素κ抑制VSV-GFP感染鸡成纤维细胞(CEF)的效果进行检测,结果显示鸡干扰素κ的抗病毒效价可以达到(1.04±0.23)×107 IU·mL-1以上。高效价鸡κ干扰素的成功表达为其在抗家禽疫病等方面提供了重要的试验数据。 相似文献
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John A. Armstrong 《Applied microbiology》1971,21(4):723-725
A technique is described for the demonstration of bacterial capsules by using montmorillonite in combination with a conventional staining method. 相似文献
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顺乌头酸酶(aconitase,Aco)是细胞内重要的铁硫蛋白酶,它催化细胞内柠檬酸经中间产物顺乌头酸生成异柠檬酸. 真核细胞中顺乌头酸酶有两种,分别定位在细胞质的顺乌头酸酶1(c-Aco)和定位在线粒体的顺乌头酸酶2(m-Aco).检测它们活性的变化能敏感地反映出细胞中能量代谢、自由基产生、铁硫簇组装及铁代谢水平的改变. 顺乌头酸酶活性的传统检测方法通常是测定细胞中总的顺乌头酸酶活性,该方法难以准确区分出c-Aco和m-Aco各自的活性变化.因此我们建立一种胶内酶活性分析法检测顺乌头酸酶活性. 该方法利用非变性电泳技术将c-Aco和m-Aco浓缩分离,通过泡染底物显色,条带颜色深浅反映了酶活性的强弱. 同时,比较了胶内酶活性分析法和分光光度法检测细胞内c-Aco和m-Aco的活性,并对比检测了过氧化氢处理细胞前后Aco活性的变化.结果显示,这两种方法均可敏感地检测出Aco的活性改变,并有广泛的细胞系实用性,但胶内酶活性分析法可区别测定c-Aco和m-Aco活性,不需繁琐的细胞质和线粒体分离,简便易行.文中介绍的线粒体分离纯化技术也为线粒体功能深入研究提供了一个快速、高效的分离纯化方法. 相似文献
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Kei Yamanaka 《Bioscience, biotechnology, and biochemistry》2013,77(6):834-839
The d-xylose isomerase activity was assayed spectrophotometrically as NADH oxidation in a coupled reaction with the d-arabitol dehydrogenase. The assay system is based on the following reactions: d-Arabitol dehydrogenase was purified from the d-sorbitol-grown cells of Agrobacterium tumefaciens. The standard assay condition is as follows: 5 μmoles of Tris-HCl buffer (pH 7.0), 0.2 μmole of MnCl2, 2 μl of reduced glutathione (25 mg/ml), 0.05 μmole of NADH, 6 units of d-arabitol dehydrogenase, 5 μmoles of d-xylose and d-xylose isomerase in a total volume of 0.30 ml. The reaction was carried out at 30°C. With the assay system, it was confirmed that d-xylose isomerase did not produce d-xylulose from d-lyxose. 相似文献
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Walter H. Traub 《Applied microbiology》1969,18(1):51-56
One of the drawbacks of the "tube dilution" method for the assay of antibiotics in human serum has been illustrated by utilizing serum-sensitive and serum-resistant strains of Escherichia coli. In the case of serum-sensitive strains, it was found that fresh serum alone may account for the same degree of inhibition and thus yield minimal inhibitory concentrations identical to those obtained with serum combined with antibiotics, that is, "simulated" serum assay specimens. This fallacy of the method is discussed with regard to those instances in which laboratories were merely to utilize the patient's own coliform organism as the test organism, or with respect to the assay of, for example, polymyxins, in which inadvertently a R(ough) and therefore, serum-sensitive strain of E. coli were to be used as the indicator organism. It is recommended that serum-resistant laboratory strains of Staphylococcus aureus or E. coli of known antibiotic susceptibility be employed as the test organisms proper in order to circumvent the inherent bactericidal activity of serum. 相似文献
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目的:通过定点突变,构建集成干扰素(IIFN/165S),以期获得高效的新型药物分子.方法:采用PCR体外定点突变技术,使集成干扰素IIFN基因的第165位密码子由CGT突变为AGT.扩增片段克隆入pET-23b表达载体,重组质粒转化大肠杆菌BL21(DE3).在LB培养基中培养,经IPTG诱导表达的IIFN/165S经包涵体变性、复性以及层析纯化后,经SDS-PAGE、Western blot和MALDI-TOF-MS分析,用WISH-VSV系统进行抗病毒活性测定同时应用流式细胞术检测细胞凋亡率.结果:IIFN/165S以包涵体形式表达.纯化后,IIFN/165S的纯度大于95%,分子量为18172,比活性(7.63±0.22)×108 IU/mg,诱导细胞凋亡率呈剂量依赖.结论:构建了IIFN/165S的表达载体,并成功地在大肠杆菌中表达,获得了高纯度高活性突变分子IIFN/165S. 相似文献
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A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the higher, unattenuated fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate consumed by renin cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate have been shown to be consistent with a simple substrate inhibition model with a substrate Km 1.5 μM at physiological pH; Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen, as reported earlier. In this report, we describe in detail the synthesis of the fluorogenic renin substrate and its application in assays of renin activity. Assay sensitivity has been evaluated by a series of enzyme dilution experiments using the continuous assay format, showing that the assay can detect renin as low as 30 ng/ml after a incubation of only 3-5 min. It was estimated that with extended incubation time (2-3 h) the assay can detect renin at 0.5 ng/ml concentration level. An automated, high throughput fluorometric renin assay has been developed for a 96-well microtiter-plate fluorescence reader, which is useful for studies of enzyme inhibitors and enzyme stability. 相似文献
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Optimization of the Interferon Assay Using Inhibition of Semliki Forest Virus-Ribonucleic Acid Synthesis 总被引:2,自引:1,他引:1
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The method for assaying chicken interferon by its inhibition of viral ribonucleic acid (RNA) synthesis was optimized for the chicken embryo fibroblast-Semliki Forest virus (SFV) system, with respect to time, multiplicity of infection, and addition of actinomycin D and (3)H-uridine. Incorporation of (3)H into viral RNA is reproducible, and amounts to 10(4) to 2 x 10(4) counts per min per uninhibited culture per 1 muCi per 6 hr. The assay may be carried out in less than 1 day and is sensitive (0.05 units/ml) and exact (+/-20% of the mean titers on different days); it can be used for purification procedures as well. 相似文献
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INTERFERON induces cellular synthesis of the antiviral protein which is probably responsible for conferring antiviral activities. Although this antiviral protein has not been isolated, indirect evidence favours a two-step mechanism in the development of cellular resistance to viruses. Taylor1 and subsequently Friedman and Sonnabend2 and Lockart3 have shown that from 0 to 4 h after the treatment of the cells with interferon, the induction of the antiviral state requires the integrity of the cellular apparatus for protein synthesis. Cassingena et al.4 have shown that in somatic mouse-monkey hybrid cells, the genes which code for the production and action of interferon are located at different chromosomal sites. 相似文献
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胰蛋白酶活性的定量测定方法 总被引:4,自引:0,他引:4
对甲苯磺酰基精氨酸甲酯(TAME)是胰蛋白酶的专一性底物. TAME经胰蛋白酶水解释放出的对甲苯磺酰基精氨酸与活性测定混合物中的NaOH反应, 导致溶液pH值的下降. 以酚红为指示剂, 通过测定555nm处光吸收值的降低可以监测pH的变化. 在0.001~0.3μg的范围内, 胰蛋白酶含量与555nm处光吸收值的降低呈线性关系. 相似文献
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Dye Uptake Assay: an Efficient and Sensitive Method for Human Interferon Titration 总被引:5,自引:1,他引:4
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Anne L. R. Pidot 《Applied microbiology》1971,22(4):671-677
Currently, human interferon (IF) assays are generally performed by plaque reduction or visual cytopathic effect methods. Both are time-consuming, subjective in interpretation, and, in the case of the latter, relatively insensitive. An adaption of the dye uptake method for human IF titration which uses foreskin-derived fibroblasts and vesicular stomatitis virus is described. This assay is reproducible and sensitive (1 unit = 3 international units). By direct comparison, however, it is somewhat less sensitive than the plaque reduction assay (1 unit = 1.1 international units). This assay is especially recommended for use in needed clinical investigations of human IF because of its technical simplicity, allowing efficient handling of large numbers of specimens. 相似文献
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Basirudeen Syed Ahamed Kabeer Rajasekaran Sikhamani Sowmya Swaminathan Venkatesan Perumal Paulkumaran Paramasivam Alamelu Raja 《PloS one》2009,4(5)