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1.
将嗜水气单胞菌(Ah)J-1株的HEC毒素和胞外蛋白酶(ECP)分别与迟缓爱德华氏菌(Et)38株的脂多糖(LPS)及脱毒多糖(PS)偶联,制成3种亚单位二联疫苗;HEC-PS、HEC-LPS。将它们与AhJ-1和Et38全菌灭活疫苗一同分别免疫小鼠,并设注射PBS对照组,检测各组小鼠体液免疫及细胞免疫水平。结果显示,用间接ELISA、溶血抑制和全菌凝集试验检出这3种亚单位疫苗的抗体水平无明显差异  相似文献   

2.
减蛋综合征病毒全基因组DNA文库的构建及物理图谱分析   总被引:1,自引:0,他引:1  
E D S V A A2 株纯化 D N A 经 Hind Ⅲ、 PstⅠ和 Sph Ⅰ水解后, 分别提取 D N A 片段并克隆至p Bluescript K S( + ) 和p G E M3 Zf ( + ) 载体, 构建了 E D S V 全基因文库。根据 E D S V D N A 片段和基因组 D N A 电泳迁移率计算, E D S V 基因组大小为329kb , 通过克隆片段酶谱鉴定, 杂交建立了 E D S V 限制性内切酶 Hind Ⅲ、 Pst Ⅰ和 Sph Ⅰ的物理图谱。  相似文献   

3.
酶标SPA在鸭病毒性肝炎免疫检测上的应用研究   总被引:3,自引:0,他引:3  
在酶标SPA(金色葡萄球菌A蛋白)的Dot Blot Immunoasay(免疫斑点试验)中,SPA能与鸭血清中IgG分子Fc段结合。这一点,在国内外尚未见报道。用酶标SPA的Dot Blot Immunoassay、Dot Blot ELISA及鸡胚中和试验,分别对DHV(鸭肝炎病毒)A毒株、及已知的I型DHVR毒株,进行交叉试验的结果一致。该试验以及病毒粒子的电镜观察,进一步表明,我们培育的优  相似文献   

4.
参照澳大利亚鸡传染性法氏囊病病毒( I B D V)00273 毒株基因组序列设计的 1 对引物,位于 V P2 高可变区两端,通过 R T P C R,扩增了 5 个 I B D V 山东分离株 V P2 基因的 607~1 080 位核苷酸片段(aa203~306)。 P C R 产物经纯化后,分别用 Dra Ⅰ、 Sac Ⅰ、 Ssp Ⅰ、 Pst Ⅰ和 Sau3 A I共 5 种限制性内切酶( R E)进行消化处理,结果 5 个分离株均为 Dra Ⅰ(- )、 Sac Ⅰ(- )和 Sau 3 A I(+ ), L C2 和 T A 株为 Ssp Ⅰ(+ ), L C1 和 J N 株为 Ssp Ⅰ(- ), L C1 和 L C2 株为 Pst Ⅰ(+ ), J N、 L L 和 T A 株为 Pst Ⅰ(- );5 个分离株的酶切图谱与美国变异株迥异,而 T A 株与日本超强毒株 9011 相类似。5 个分离株与现用疫苗株对比,至少在 V P2 可变区内存在着一定的差异,这可能是 I B D V 接种免疫鸡群仍然暴发 I B D 的原因之一。  相似文献   

5.
在酶标SPA(金色葡萄球菌A蛋白)的DotBlotImmunoasay(免疫斑点试验)中,SPA能与鸭血清中IgG分子Fc段结合。这一点,在国内外尚未见报道。用酶标SPA的DotBlotImmunoassay、DotBlotELISA及鸡胚中和试验,分别对DHV(鸭肝炎病毒)A毒株、及已知的I型DHVR毒株,进行交叉试验的结果一致。该试验以及病毒粒子的电镜观察,进一步表明,我们培育的优良鸭肝炎弱毒株A66确系I型DHV。  相似文献   

6.
抗新城疫病毒糖蛋白单克隆抗体的研制及其特异性分析   总被引:5,自引:0,他引:5  
应用纯化的新城疫病毒F48E9株免疫BALB/C小鼠,取脾细胞与SP2/P骨髓瘤细胞融合,经间接ELISA法检测筛选到42株分泌抗HDV单克隆抗体的杂交瘤细胞株。并对各株单克隆抗体的免疫球蛋白亚类,血凝抑制效价,溶血抑制效应ELISA效价及中和效价加 测定,结合单克隆抗体的Westerb blot反应结果出抗NDV HN蛋白11株,抗F蛋白20株。  相似文献   

7.
同一条件下研究比较杜洛克(D)与A、B、C三个新品系的杂交猪的肉用性能。结果显示,在25~100kg阶段,DA、DB、DC的生长速度分别为75732、71881、68667g/d。料肉比依次为358、354、370。屠宰测定,DA、DB的胴体瘦肉率比DC分别高751%(P<005)、1338%(P<001),脂肪率DC比DA、DB分别高1484%(P<005)和3115%(P<001),DA和DB无显著性差异。其他胴体性状水平无显著性差异。DA、DC的肉质性能良好,DB表现轻度的PSE肉。DA、DB90kg适宜上市,DC100kg适宜上市。  相似文献   

8.
本研究利用10%SDS-PAGE及Western-Blotting技术鉴定3株不同的减蛋综合征病毒(贵州株HS-1、南京分离毒GC2、国际标准毒AV-127)的蛋白抗原性,结果表明,3株毒的蛋白多肽分子量均在106-12kD范围,一各条多肽的组成上略有差异;它们都具有两条相同的免疫原性多肽,分子量分别为106和100KD。同时,运用EcoRⅠ,HindⅢ,PstⅠ和SmaⅠ等5种限制性内切酶进行了酶切分析,证实3株EDS病毒用PstⅠ和SmsⅠ酶切的片段大小及长度略有不同,而其它3种酶的酶切图谱完全相同。  相似文献   

9.
本研究主要对猪生殖呼吸道综合征( P R R S) 国内分离毒株( C H Ia) 进行了病毒纯化及其结构蛋白的分析。选用聚乙二醇( P E G6000) 沉淀和差速离心法粗提了经 Marc145 细胞系增殖的猪生殖呼吸道综合征病毒( P R R S V) 。并采用非线性蔗糖密度梯度离心法纯化了 P R R S V, 经免疫电镜观察纯化病毒, 发现有较多的胶体金颗粒吸附在直径为42 ~78nm 的病毒粒子周围。经 Dot E L I S A 测定纯化病毒的滴度可达1 1600 以上, 并利用 S D S P A G E 和 Westernblot 两种方法对国内分离毒株( C H Ia) 与欧洲型( Lelystad) 、美洲型( V R2332) 等参考毒株的病毒结构蛋白组成和部分抗原特性进行了初步的研究。测得国内分离毒株主要结构蛋白的分子量为145 K D,192 K D 和263 K D, 并均能被 P R R S V 的高免血清识别, 这与国外的同类报道很相似。  相似文献   

10.
含绿脓杆菌外毒素A受体结合区重组蛋白的纯化   总被引:5,自引:3,他引:2  
将表达含绿脓杆菌外毒素(PEA)受体结合区基因的质粒pET-EAB转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达了含PEA受体结合区的重组蛋白(称PE34)。PE34主要以包涵体形式存在于大肠杆菌中,用溶菌酶-脱氧胆酸钠法结合超声波裂解法破碎细菌,离心制备包涵体。用2mol/L脲洗涤包涵体后,包涵体纯度可达75%,在8mol/L脲存在条件下,SephacrylS-200凝胶滤过,DEAE-SepharoseFastFlow离子交换层析纯化,获得SDS-PAGE1条带的PE34,纯度达95.8%,得率为24.5%。  相似文献   

11.
迟缓爱德华氏菌检验程序的研究   总被引:10,自引:0,他引:10  
本试验研究了迟缓德华氏菌(Edwardsiellatrada,Et)的细菌学及致病因子检测,确定了相应检测指标,制定了检验程序。对不同来源的Et作分离及生化鉴定,确定10项生化鉴定指标。用三种方法即平板法(PlateAssay,PA)、接触法(ContactHemolysis,CH)和上清法(SunernatantAs-say,SA)检测Et溶血素,阳性率依次为75%、75%、57.14%。28株Et中,18/26的胞外产物(Extra-cellularProducts,ECP)对HEP-2细胞有细胞毒性,17/28菌株有侵袭力。用ATCC15947株ECP的抗血清进行Det-ELISA,检测阳性率为19/26(73.08%)。  相似文献   

12.
The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P greater than 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

13.
Three gut lactobacilli from piglets (Lactobacillus plantarum L 5, Lactobacillus paracasei L 81, Lactobacillus fermentum L 670) and Lactobacillus casei subsp. pseudoplantarum L.c.) from a calf were examined by microtitre plate binding assay for their lectin-like binding activity after their cultivation on Rogosa agar and in MRS broth. Three ECM (extracellular matrix) molecules (fetuin, porcine fibronectin and porcine mucin) were selected for this assay. Additionally, the effect of heparin on the binding of these three ECM molecules by Lactobacillus strains in microtitre plates was tested. Moreover, haemagglutination tests with pig, cattle, sheep, and hen erythrocytes were performed. However, none of the four Lactobacillus strains examined did react with any of the erythrocytes tested. The differences between individual strains were observed in their binding to immobilised ECM molecules. The best adherent was the Lactobacillus plantarum L5, however, the other three strains showed also good ECM binding. With regard to an influence of cultivation medium on lectin-like binding activity, binding of all ECM molecules was expressed in Lactobacillus paracasei L 81 to significantly higher degree (P < 0.001) after cultivation on Rogosa agar than in MRS broth. Similarly, strains Lactobacillus fermentum L 670 and Lactobacillus casei subsp. pseudoplantarum L.c. displayed significantly higher (P < 0.001) binding of fibronectin and mucin after growth on Rogosa agar in comparison with MRS broth cultivation. However, no significant (on fetuin and fibronectin binding) or opposite effect (on mucin binding) of cultivation medium was observed in Lactobacillus plantarum L 5 strain. The influence of cultivation medium on fetuin binding by Lactobacillus fermentum L 670 was also not significant while Lactobacillus casei subsp. pseudoplantarum L.c. bound fetuin significantly better (P < 0.01) after growth on Rogosa agar. Heparin pretreatment increased the binding of the ECM molecules by the Lactobacillus fermentum L 670 strain significantly (P < 0.001 or P < 0.05) with the exception of porcine fibronectin when the strain was cultivated in MRS broth. This result is important especially in the connection with the previous observations that heparin decreased ECM binding of enteropathogens as staphylococci or clinical enterococcal isolates. Following up on some earlier strain characteristics, these results indicate that the selected lactobacilli are probably suitable for probiotic purposes.  相似文献   

14.
Superoxide dismutases of virulent and avirulent strains of Brucella abortus   总被引:7,自引:0,他引:7  
Extracts of Brucella abortus strains 2308,RB51,45/20 and ST 19 had no significant differences in superoxide dismutase (SOD) activity as measured by the epinephrine assay. These B. abortus strains represent smooth, intermediate and rough colony forms. SOD activity was inhibited 60 to 75% by 2 mM KCN and suggests the presence of Cu/Zn SOD. The SOD activities were similar when the strains were grown in trypticase soy broth containing either 0.5% glucose or erythritol. There were two distinct SOD activity bands in native polyacrylamide gel electrophoresis with identical mobilities for each of the strains. When the native gel was stained for SOD activities in the presence of 2 mM KCN, the SOD band that co-migrated with the bovine erythrocyte Cu/Zn SOD activity disappeared. The band of SOD activity that migrated similar to E. coli iron SOD activity was unaffected by KCN. There were no significant differences in either the total SOD or Cu/Zn SOD activities among the strains. As the Brucella strains represent ranges of virulence, it is difficult to associate any primary role for SOD as a virulence factor.  相似文献   

15.
OBJECTIVE: To assess effects on functions of peripheral blood mononuclear cells (PBMC) obtained from ewes for each of several fatty acids represented in ovine plasma at concentrations mimicking those of ketotic or healthy ewes. SAMPLE POPULATION: Blood samples obtained from 6 Sardinian ewes. PROCEDURE: The PBMC were cultured in media that contained oleic (OA), palmitic (PA), stearic (SA), linoleic (LA), or palmitoleic (POA) acid at concentrations similar to those of ketotic or healthy ewes. Synthesis of DNA was stimulated by use of concanavalin A or pokeweed mitogen (PWM). Secretion of IgM was stimulated by use of PWM. RESULTS: High concentrations (900, 450, and 225 micromol/L) of OA significantly inhibited DNA synthesis and IgM secretion of PBMC. Conversely, low concentrations (56 or 28 micromol/L) of OA significantly enhanced DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 375, or 18.7 micromol/L) and SA (300, 150, or 75 micromol/L) significantly inhibited DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 375, or 18.7 micromol/L) and SA (300, 150, 75, or 38 micromol/L) also significantly inhibited IgM secretion of PBMC. None of the concentrations of LA and POA affected PBMC functions. CONCLUSION AND CLINICAL RELEVANCE: Impaired immunoresponsiveness of ketotic ewes is likely associated with an increase of plasma concentrations of OA, PA, or SA and not with that of LA or POA. At physiologic concentrations, single fatty acids are likely to participate in modulation of immunoresponsiveness by exerting suppressive or stimulatory effects on immune cells.  相似文献   

16.
Hemolytic complement levels in 30 duplicate samples of normal bovine sera were determined by a tube titration method (CH50) and by a radial hemolysis in gel assay. Significant correlation was not observed between the values obtained by the 2 tests. This lack of correlation could be a result of considerable error observed in values obtained for duplicate samples in the CH50 method or to the relative insensitivity of the hemolysis in gel test. The hemolytic reaction in gel was found to depend on Mg2+, thus raising questions (i) about its validity in determining total hemolytic complement or (ii) about bovine C1q depending on Mg2+ rather than Ca2+ for binding to immune complexes.  相似文献   

17.
Loop-mediated isothermal amplification (LAMP) assay is a simple, rapid and specific detection method and has been used for detection and identification of different Campylobacter species. In this study, we develop a LAMP assay specific for detection of a particular clone (clone SA) of Campylobacter jejuni, associated with the vast majority of recent sheep abortions in the U.S. Using a set of specific primers for C. jejuni IA3902 (a clone SA isolate) and genomic DNA or boiled cell extract as template, the target DNA was amplified at 63 °C for 50 min in a water bath. A positive reaction was identified visually as white precipitate or fluorescence under UV, and confirmed by gel electrophoresis. Detection limit of the assay was comparable to that of conventional PCR. The LAMP was shown to be specific for detection of clone SA when tested on a number of C. jejuni strains of different genetic backgrounds. Applicability of the LAMP assay for specific detection of clone SA was demonstrated in animal tissues experimentally infected with IA3902 or genetically diverse C. jejuni strains. Since clone SA is the predominant cause of sheep abortions in the U.S. and is a zoonotic pathogen, the LAMP assay may be a valuable detection tool in future epidemiological studies.  相似文献   

18.
本研究旨在对从鸡肠道里分离到的2种抗菌肽进行生物学性质研究.从鸡盲肠内容物中分离得到2株菌,通过生理生化法和16S rRNA测序分析进行鉴定.采用牛津杯法对抗菌肽的抗菌谱进行研究;通过调节培养基的碳源、氮源、及初始pH研究培养条件对抗菌肽产量的影响.分离鸡盲肠内容物得到2株菌,鉴定为植物乳酸杆菌CLP29和粪肠球菌CLE34,它们可以产生2个蛋白酶敏感的抗菌肽(植物乳酸菌素CLP29,粪肠球菌素CLE34,3.5 ku).在MRS(Mann Rogosa Sharpe)培养基中,当初始pH为5.0、6.0和7.0,且培养温度为37℃时,2种抗菌肽的抗菌活性均达到最大值,为6 400 AU/mL.碳源和氮源均影响植物乳酸杆菌素CLP29和粪肠球菌素CLE34的产量.可溶性淀粉可以刺激植物乳酸杆菌素CLP29的表达,但是当氮源为柠檬酸铵或蛋白胨加牛肉浸膏时,植物乳酸杆菌素CLP29产量则大大降低.对粪肠球菌素CLE34而言,碳源为半乳糖时,产量可达到最大(6 400 AU/mL),但是当碳源为可溶性淀粉或蔗糖时,产量则降低50%~70%.综上,本研究从鸡的肠道中成功地分离得到可产生抗菌肽的2株菌,且通过改变培养基组成和条件实现对抗菌肽产量的调节.  相似文献   

19.
1. The distribution of parvalbumin (PA), which functions as a relaxing factor in the skeletal muscles, was examined in slow anterior latissimus dorsi (ALD), fast posterior latissimus dorsi (PLD), mixed sartorius (SA), pectoralis superficialis (PS) and pectoralis profundus (PP) muscles from chickens.

2. The biochemical characteristics of these muscles were confirmed by the assay of total lactate dehydrogenase (LDH) activity as well as the LDH isozymes for anaerobic metabolism, and by the photometrical analysis of myoglobin for aerobic metabolism.

3. PA in individual muscles was determined by a sandwich ELISA and was demonstrated by 2‐dimensional polyacrylamide‐gel electrophoresis.

4. Because of poor myoglobin and higher LDH activity or M‐type isozyme pattern, the PLD was confirmed as containing primarily fast‐twitch glycolytic and oxidative‐glycolytic (FG/FOG) fibres, while the SA was shown to be composed mosdy of FOG fibres because of the highest myoglobin content and the intermediate LDH activity or H and M‐type isozyme pattern. PA content was high and variable in both PLD and SA.

5. PA was undetectable in the ALD which appears to contain exclusively slow‐tonic (ST) fibres, being verified by its myoglobin‐rich nature and lowest LDH activity or predominant H‐type isozyme characteristics. PA was absent from the PS and PP, which probably contain predominantly FG fibres because of negligible amounts of myoglobin and the highest LDH activities or M‐type isozyme pattern.  相似文献   


20.
A total of 77 field isolates and 15 reference strains of the porcine respiratory pathogen Actinobacillus pleuropneumoniae were tested for their ability to form biofilms in a polystyrene microtiter plate assay. More than half of all field isolates, which included strains representing serotypes 1, 5 and 7, but only two reference strains (serotypes 5B and 11) exhibited biofilm formation. Strains that formed biofilms in microtiter plates also formed thick biofilms at the air-liquid interface when cultured in glass tubes with agitation. The biofilm formation phenotype was maintained indefinitely when cultures were passaged on agar but was lost after one or two passages in broth. Our findings indicate that biofilm formation is a prevalent phenotype among A. pleuropneumoniae field isolates, and that this phenotype may have been previously overlooked because of its tendency to be lost upon subculturing in broth. Biofilm formation may have relevance to the colonization, pathogenesis and transmission of this bacterium.  相似文献   

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