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产单核细胞李斯特菌溶血素的纯化 总被引：1，自引：0，他引：1
本文旨在探讨产单核细胞李斯特茵溶血素（LLO）的纯化方法。将产单核细胞李斯特茵（LM）接种于BHI液体培养基中培养,得到含LLO的培养液上清（LLO1）,再经70％饱和硫酸铵沉淀、透析,得到溶血素粗提物（LLO2）,LLO2经凝胶过滤层析,聚乙二醇浓缩,得到活性峰收集液（LLO3）。通过以上三步的纯化,溶血素的比活提高160倍,纯化蛋白在SDS-PAGE中呈现一条带,达电泳级纯度。 相似文献
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对致羔羊脑炎肠球菌E129-3菌株溶血素进行检测分析，并利用光学显微镜和电子显微镜观察其对兔红细胞的作用，探讨了肠球菌溶血素的特性及溶血类型。结果表明，小白鼠、兔、犬红细胞对该溶血素最敏感，牛红细胞不敏感。该溶血素可以被DTT、Ca^2＋活化，被蛋白酶K、EDTA、Zn^2＋、Cu^2＋抑制。在培养后2h，细胞浆内着色不一致或密度降低，5h时红细胞数量明显减少，细胞膜较完整。提示，肠球菌溶血素为成孔蛋白类溶血素。 相似文献
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嗜水气单胞菌 β-hemA 重组菌的优化培养及其生长曲线的测定 总被引：3，自引：0，他引：3
用正交试验探讨温度、培养基、时间三个因素对嗜水气单胞菌β-hemA重组菌E.coliDH5α(PCB)基因产物表达量的影响,通过优化培养,选择最佳培养条件,显著提高了β的溶血素基因(β-hemolysisgene,β-hemA)产物的产量。实验结果表明:该重组菌株在100mlTSB(添加0.05%酵母浸提物,1.8μg/mlZnCl2),37℃培养30h收获,其毒素蛋白含量为3258μg,毒素溶血价为17.44hu/μg,而在LB(100ml)和营养肉汤(100ml)中最高蛋白量和溶血活性分别为1260μg,14hu/μg和1199μg,8.22hu/μg。所选择的三个因子中对毒素蛋白质含量影响最大的是培养基,其次是温度;而对溶血素影响最大的则是温度,其次为时间。最佳生长曲线测定结果表明,在培养基预热至37℃的情况下,PCB6h能进入对数期。 相似文献
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本文旨在探究黄酮类化合物槲皮素对金黄色葡萄球菌α-溶血素所引起的大鼠肺微血管内皮细胞(RPMVECs)凋亡的影响。采用贴块法培养原代细胞后用磁极纯化法选获取纯化的RPMVECs,并通过CD31免疫荧光进行鉴定。通过WST-1检测α-溶血素对RPMVECs的细胞毒性,筛选作用浓度。将槲皮素与α-溶血素共同作用RPMVECs 24 h后,通过流式细胞术检测细胞凋亡率。结果表明采用磁极纯化法可得到CD31阳性率高的RPMVECs。2μg/mLα-溶血素对RPMVECs具有毒性,并可诱导RPMVECs凋亡。槲皮素可以抑制金黄色葡萄球菌α-溶血素所诱导的RPMVECs凋亡,对细胞具有一定保护作用。 相似文献
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通过乙醇回流提取获得中草药连翘粗提物,利用MIC值测定、溶血活性测定、免疫印迹、RT-PCR、LDH及live/dead等试验方法研究连翘提取物对金黄色葡萄球菌α-溶血素分泌的影响.结果表明:连翘提取物对金黄色葡萄球菌的最小抑菌质量浓度(MIC)均大于2 048 mg/L,表明连翘提取物不影响金黄色葡萄球菌生长.连翘提取物在16～128 mg/L时,抑制了金黄色葡萄球菌α-溶血素的分泌,而且这种抑制作用呈剂量依赖性.研究结果提示连翘提取物可作为一种潜在的抗金黄色葡萄球菌感染药物,可进一步开发. 相似文献
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胸膜肺料放线杆菌溶血毒素特性鉴定及免疫原性研究 总被引：4，自引：0，他引：4
本文报道了胸膜肺炎防线杆菌（APP）血清型7型菌株深－8株分泌的溶血素。研究证明该株至少可分泌两种溶血素，一种为热稳定的溶血素，另一种为热不稳定的溶血素。本研究没有对热稳定的溶血素进行提纯分析，而对热不稳定的溶血素经过盐析及凝胶过滤层析提纯后，进行SDS－PAGE分析，表明该溶血素为分子量65kDa的蛋白。本研究利用纯化的65kDa的溶血素蛋白与APP所有型的阳性血清做交叉中和实验和琼脂扩散实验及动物实验，证明该溶血素蛋白具有一定的免疫原性及保护力，但其诱导的免疫反应对同源菌株的攻击只能提供部分保护。 相似文献
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猪链球菌2型作为一种人兽共患病病原,日益受到关注。而溶血素是其分泌的外毒素,是公认的重要毒力因子之一,具有良好的免疫原性。本试验通过PCR方法获得猪链球菌2型野生型溶血素基因sly和463、464双点突变的溶血素突变体基因slym。将sly和slym克隆至表达载体,构建了2个重组载体并在大肠杆菌中表达了野生型溶血素rSLY和突变型溶血素rSLYm。经过蛋白杂交试验,证明表达的rSLY和rSLYm与提取的猪链球菌的天然野生型SLY分子量完全一致。以提取的野生型SLY为对照,通过溶血试验证明,突变型溶血素rSLYm失去了溶血活性;通过接种PK15、RK13、SUVEC细胞单层,证明突变型溶血素rSLYm失去细胞毒性;通过小鼠试验,证明突变型SLYm对小鼠没有毒力。本试验通过溶血试验、细胞接种和小鼠实验,证明双点突变灭活了野生型溶血素的溶血活性、细胞毒性和小鼠毒力。该溶血素突变体经免疫实验证实后,可作为猪链球菌2型亚单位疫苗的候选抗原。 相似文献
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Haemolytic activities of Trichophyton (T.) mentagrophytes were detected and characterized by qualitative and quantitative assays. On Columbia agar supplemented with blood from horses, cattle or sheep, T. mentagrophytes expressed a strong zone of complete haemolysis. No haemolytic activities could be detected in the closely related T. verrucosum var. ochraceum. The same results were obtained after cultivation of the fungi on sterile cellulose acetate filters placed on the surface on Columbia blood agar. After removal of the filter, complete haemolysis was detected below the colony of T. mentagrophytes. A soluble haemolysin from culture supernatant of this strain was isolated and partially purified. Specific haemolytic activity per mg protein was enriched 2.6-fold in filtrate F(1), a fraction obtained as filtrate after filtration through 3kDa cut-off membranes. The partially purified haemolysin was neither affected by proteinase K treatment, nor by high and low temperatures, suggesting that it represents a small peptide haemolysin. Accordingly, in a commercial enzymatic activity test only the crude culture filtrate, but none of the subsequent purification fractions showed reactivity. Evaluation of the specificity of the haemolysin using erythrocytes from different mammalian species revealed that sensitivity was highest to those of equines, followed by erythrocytes from sheep, cattle, swine, dogs and humans. None of the erythrocytes was lysed by filtrate F(1) from T. verrucosum var. ochraceum. Furthermore, different eukaryotic cell lines from different species were tested in their sensitivity to cytolytic activities of the haemolysin, but no membrane damage could be detected. 相似文献
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Endothelial cytotoxicity of Actinobacillus pleuropneumoniae 总被引：5，自引：0，他引：5
S Serebrin S Rosendal A Valdivieso-Garcia P B Little 《Research in veterinary science》1991,50(1):18-22
The cytotoxicity of Actinobacillus pleuropneumoniae serotype 1 strain CM5 for porcine and bovine endothelial cells in vitro, was dose-dependent. This strain and its attenuated and avirulent substrain CM5A were equally cytotoxic. The cytotoxicity observed during five hours of exposure of endothelial cells to bacterial products was abolished if the bacteria were inactivated by heat or sonication. Exposure of the endothelial cells for five hours to 100 and 200 micrograms of purified lipopolysaccharide resulted in a partial cytotoxicity only, which was not enhanced in the presence of fresh guinea pig serum. The cytotoxicity of viable bacteria could be neutralised by a polyclonal rabbit antiserum to the purified 104kD haemolysin. A bacteria-free supernate of a culture of strain CM5 had both haemolytic and cytotoxic activity. The haemolytic activity could be neutralised completely by the anti-serum to the 104kD haemolysin, whereas the cytotoxic activity was only partially neutralisable. Hence A pleuropneumoniae is cytotoxic for endothelial cells and this cytotoxicity is possibly mediated by the 104kD haemolysin. 相似文献
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家蚕γ-谷氨酰环化转移酶的纯化及其性质的研究 总被引：3，自引：0，他引：3
首次从家蚕(Bombyx mori L.)蛹体脂肪织织及真皮细胞分离得到催化丫-L-谷氨酰-2-L-氨基丁酸反应生成吡咯烷酮羧酸和2-L-氨基丁酸的Y-谷氨酰环化转移酶。并采用硫酸铵沉淀、葡聚糖G-75柱层析、DEAE-纤维素DE_(32)柱层析和羟基磷灰石柱层析等步骤,将该酶提纯了1,285倍。高度纯化后的酶,对Y-L-谷氨酰-2-L-氨基丁酸具有最适pH7.2-7.4,最适酶反应温度为47℃,米氏常数(Km)为0.04M。在分离纯化过程中未发现蚕体内有该酶的同功酶存在,纯化后的酶溶解于pH8.0、0.01M的Tris—HCl缓冲掖中,-30℃经过一个月没有发现明显的失活现象,但在4℃中一个月后丧失活性76％。 相似文献
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K S Ghenghesh S S Abeid M M Jaber S A Ben-Taher 《Comparative immunology, microbiology and infectious diseases》1999,22(3):175-179
Rectal swabs from 120 domestic dogs and 15 domestic cats were examined for Aeromonas species using alkaline peptone water (pH 8.6) as the enrichment medium and blood agar containing 15 mg/l ampicillin as the plating medium. Aeromonads were isolated from 13 (10.8%) dogs and from 1 (6.7%) cat. Of the 14 aeromonads isolated in the present study only 9 were available for speciation and testing in the haemolysin assay. Of these 5 were A. sobria (including one from a cat), 2 were A. hydrophila and 2 were A. caviae. Six were positive in the haemolysin assay; 4 A. sobria (one from a cat) and 2 A. hydrophila. The presence of haemolysin producing-Aeromonas species in the faeces of domestic dogs and cats may pose a public health problem for humans who come into contact with such animals. 相似文献
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The thermostability of proteases from virulent and benign strains of Bacteroides nodosus 总被引：1，自引：0，他引：1
Protease enzymes, produced by Bacteroides nodosus strains isolated from animals with virulent and benign forms of ovine footrot, were partially purified by ultra-filtration, ion exchange chromatography and gel permeation chromatography. Each enzyme had a similar pH optimum, was inhibited by phenylmethylsulfonyl fluoride (PMSF), ethylene diamine tetraacetic acid (EDTA) and ethyleneglycot-bis-aminoethylether-N,N-tetraacetic acid (EGTA), but was not inhibited by 1,10-phenanthroline. The results suggest that these enzymes are serine proteases that require divalent cations for activity. The enzymes could be distinguished by their differential temperature stability and differing susceptibility to irreversible inactivation by EDTA. Both enzymes were stabilised by incubation in the presence of Ca2+, but the enzyme purified from the virulent isolate required less Ca2+ for maximum stability. These results suggest that the differential thermostability of the protease activity detected in virulence tests is an intrinsic property of the protease enzymes. 相似文献
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Sixty-eight methicillin-resistant Staphylococcus aureus strains were isolated from mastitis milk samples originating from 20 Belgian dairyherds. All these strains appeared to be representatives of one single strain which was probably of human origin. Evidence is presented indicating a rapid in vivo evolutionary change in this strain. The following characteristics were found to be variable: the production of beta haemolysin inversely connected with fibrinolysin (staphylokinase) activity; the production of lipase, enterotoxin B and delta haemolysin; the resistance to neomycin, chloramphenicol, tetracyclines, methicillin and spectinomycin associated with constitutive or inducible macrolide resistance. 相似文献
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A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group. 相似文献
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猪链球菌血清2型JX02株溶血素基因的克隆及序列分析 总被引：3，自引：0，他引：3
采用特异性引物对猪链球菌2型(Streptococus suis type 2)JX02菌株进行PCR扩增，将扩增到的溶血素基因克隆到pMDl8-T质粒栽体中，经鉴定后测序。结果显示，扩增的JX02株溶血素基因长度为1494bp，与P1／7株和US-1933株等进行序列比较，核苷酸同源性为100％和99．7％，氨基酸序列同源性为100％和99．8％。 相似文献
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R D Pearce J W Callahan A Novak P B Little J T Clarke 《The British veterinary journal》1990,146(3):270-280
Lysosomal beta-D-mannosidase (EC 3.2.1.25) was purified 6900-fold from normal goat kidney by serial Concanavalin A-Sepharose 4B and Red A dye ligand affinity chromatography, followed by anion exchange and molecular sieve high performance liquid chromatography. The relative molecular mass of the enzyme was estimated by molecular sieving to be 79,000 +/- 3000. The apparent Km for the synthetic substrate, 4-methylumbelliferyl-beta-D-mannopyranoside, was 2.3-2.8 mM and the sharp, unimodal pH optimum was 5.5. Enzyme activity was inhibited by Hg2+, Zn2+, Ag+, Co2+ and the thiol reactive agent N-ethylmaleimide. The mannose derivatives p-nitrophenyl-beta-D- thiomannopyranoside and p-aminophenyl-beta-D-thiomannopyranoside inhibited enzyme activity and may be of use as immobilized ligands in future attempts to purify beta-D-mannosidase by specific affinity chromatography. 相似文献