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动物抗菌肽天蚕素(cecropin)因具有抗菌谱广、活性较强、不易产生耐药性等特点而成为新型抗菌剂开发的材料。为了探讨通过原核表达的途径高效获取天然cecropin,以重叠PCR方法分别人工合成家蚕(Bombyx mori)和果蝇(Drosophilamelanogaster)的抗菌肽cecropin B基因(CecB2)并克隆至T载体,测序确认后分别克隆至pET-32a载体,转化至E.coli BL21(DE3)宿主菌进行表达。通过SDS-PAGE和Western blotting检测到2种目的蛋白均得到表达,但表达量存在差异,Bradford法测定纯化后的BmCecB2融合蛋白和DmCecB2融合蛋白的质量浓度分别为0.6、2.0 mg/mL。经Ni-NTA亲和层析纯化和透析处理后的2种产物的抗菌活性存在明显差异:DmCecB2和BmCecB2的质量浓度为10 mg/mL时,对E.coli DH5α的抑菌圈直径分别为15、8 mm左右,最小抑菌浓度分别为0.05、0.10 mg/mL。以上结果说明DmCecbB2的原核表达效率及表达产物的抑菌活性均明显高于BmCecB2。用生物信息学方法分析家蚕和果蝇的CecB2蛋白在结构上存在差异,这可能是导致2种产物活性不同的主要原因。 相似文献
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通过高效液相色谱法,对光滑爪蟾皮肤分泌物进行分离纯化,得到具有抗菌活性的光滑爪蟾皮肤抗菌肽。通过最小抑菌浓度（MIC）试验、溶血活性试验及癌细胞抑制试验,对其体外生物活性进行研究。结果表明,经过纯化得到2个纯度高且具有活性的抗菌肽（AMP1和AMP2）,质谱鉴定结果得出其相对分子质量分别为1 082.78和2237.34。MIC结果表明,AMP1对S.aureus ATCC25923、Escherichia coli ATCC25922的MIC值分别为23.63、8.71mg/L,对MRSA无抑菌作用。AMP2对S.aureus ATCC25923,Escherichia coli ATCC25922、MRSA的MIC值分别为4.94、10.7、86.6mg/L。溶血试验显示AMP1,AMP2具有极弱溶血活性,且对癌细胞SW480具有抑制作用。 相似文献
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《动物医学进展》2021,42(10)
为探讨地榆不同部位提取物对多重耐药大肠埃希氏菌(MDR E.coli)体外抑菌效果及抑菌机制,通过系统溶剂提取法萃取制备地榆石油醚、乙酸乙酯、正丁醇和水部位提取物,用微量肉汤稀释法检测各部位提取物对MDR E.coli的抑菌活性,筛选最佳抑菌活性部位提取物,并通过对MDR E.coli的细胞壁、细胞膜、菌体总蛋白合成的影响探讨抑菌机制。结果表明,地榆正丁醇部位提取物对MDR E.coli的抑制作用最强,其最小抑菌浓度(MIC)为3.125 mg/mL,在浓度为MIC和0.5×MIC时作用菌体12 h, MDR E.coli菌量分别为(2.37±0.59)×10~(6 )CFU/mL和(2.05±0.57)×10~8 CFU/mL;培养基中碱性磷酸酶(AKP)活性分别为(1.498±0.023)U/L和(0.768±0.088)U/L;在260 nm处培养基上清液OD值分别为0.363±0.003和0.168±0.009,均极显著高于对照组(P0.001);菌体总蛋白条带出现明显缺失。地榆正丁醇部位提取物对MDR E.coli的抑菌作用最强,其作用机制是通过破坏菌体细胞壁、增大菌体细胞膜通透性,干扰细菌总蛋白合成抑制细菌的生长。 相似文献
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天然抗菌肽具有较强的杀菌能力,但其较高的细胞毒性会使肽的细胞选择性降低。为了提高抗菌肽的选择特异性,本研究拟探讨不同疏水性氨基酸对α-螺旋抗菌肽生物学活性的影响及其抑菌机制。笔者采用α-螺旋肽GRX_2RX_3RX_2RG作为模板,分别以疏水性氨基酸色氨酸(Try,W)、苯丙氨酸(Phe,F)、缬氨酸(Val,V)、丙氨酸(Ala,A)、亮氨酸(Leu,L)和异亮氨酸(Ile,I)来替换X位置,得到了一系列富含疏水氨基酸的肽GW、GF、GV、GI、GA和GL。本研究检测了抗菌肽的二级结构、溶血活性、抑菌活性、盐离子活性,并对其抑菌的作用机制进行了研究。结果表明:通过CD光谱试验检测出GF、GI、GA和GL在细胞膜模拟环境中均表现出典型的α螺旋结构,而GV只在TFE中呈现α螺旋结构;溶血试验结果表明,GV和GA在浓度为128μmol·L-1未表现出溶血活性,而其他肽均表现出较高的溶血活性;抑菌试验发现GV的最小抑菌浓度(MIC)的几何平均值为3.7μmol·L-1,并具有最高的治疗指数(TI);盐离子稳定性试验表明,在NH_4^+、Zn2+和Fe3+中GV对大肠杆菌25922(E.coli ATCC 25922)的抑菌能力较稳定。进一步通过扫描电镜和内膜通透性试验对抗菌肽GV的抑菌机制进行分析,结果观察到GV能够通过穿透E.coli ATCC 25922和金黄色葡萄球菌29213(S.aureus ATCC 29213)促使细胞内容物流出而导致细菌死亡,以及通过时间和剂量依赖的方式穿透细菌内膜。综合以上结果,富含Val的GV具有较高的细胞选择性和成为高效抗菌药物的发展潜力。 相似文献
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为了解在亚抑菌浓度单一药物的持续诱导下,鸡源大肠杆菌(E.coli)对临床常见药物的耐药表型变化,本实验采用亚抑菌浓度的头孢曲松或头孢噻肟持续诱导培养LG30鸡源E.coli和O78标准菌至30代,并采用微量稀释法检测各不同诱导代次的菌株对常见药物的耐药表型.结果表明在亚抑菌浓度的头孢曲松或头孢噻肟持续诱导培养下,受试菌对药物的敏感性持续下降,当诱导至20代,各诱导菌已成为多重耐药菌株,继续诱导至30代,各诱导菌对药物的耐药程度加重,但各药的最小抑菌浓度值升高速率变慢.表明细菌在单一药物诱导下可以较快进化为多重耐药菌. 相似文献
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抗菌肽(Antibacterial peptide,AMP)又叫宿主防御肽(host defense peptides),普遍存在于真核和原核生物体内,参与机体的天然免疫.抗菌肽因为其显著的优点,成为替代抗生素研究的一个靶标.但是抗菌肽在应用过程中遇到了一个瓶颈问题,即原核表达系统表达蛋白的无活性及真核表达系统的不稳定性,这也制约了抗菌肽的工业化开发应用.因此,筛选自身能分泌抗菌肽的微生物,利用其自身性能稳定、易培养的优点来生产抗菌肽成为研究热点.本研究通过常规培养方法从家蚕中肠中筛选出好氧菌和兼性厌氧菌,通过观察菌落形态、细菌形态以及染色反应鉴别出不同的细菌.通过超声波破碎细胞提取细胞内容物,进行抑菌实验.结果显示中肠优势菌杆状和球状细菌的分泌蛋白对供试菌均无抑菌活性. 相似文献
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《畜牧兽医学报》2017,(12)
为了研究噬菌体Bp7穿孔素holin的特征及其对细菌的抑制作用,根据噬菌体Bp7的基因序列,设计引物PCR扩增穿孔素holin基因,并对其序列进行比对和分析。构建holin原核重组表达质粒pCold-holin,在大肠杆菌BL21中诱导表达,摸索最佳表达条件。亲和层析法纯化蛋白质,并对其抑菌活性进行测定。结果表明,噬菌体Bp7的holin蛋白含有一个跨膜区,属于Phage-holin-T家族;在E.coli BL21表达的重组holin蛋白以可溶性形式存在。表达菌E.coli BL21浊度测定试验结果表明holin蛋白的表达在4h内能够显著抑制表达菌E.coli BL21的增殖,活菌数减少,菌体出现凝集现象。研究结果提示噬菌体Bp7的holin蛋白具有抑菌活性,这将为进一步研究穿孔素的抑菌机制奠定基础。 相似文献
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Huguet A Savary G Bobillier E Lebreton Y Le Huërou-Luron I 《Journal of animal science》2006,84(11):2965-2972
The objective of this study was to determine the influence of the level of feed intake and a 2-d feed restriction period on the postweaning adaptation of pancreatic exocrine secretions. At 33 d of age, 18 piglets fitted with 2 permanent catheters (for pancreatic juice collection and reintroduction) were weaned and allocated to 1 of the following 2 dietary treatments for 5 d: restricted feed allocation (restricted) or gradually increasing feed allocation (control). Pancreatic juice was collected daily during both basal and prandial periods. The basal period was defined as the period from 1400 to 1700 h (i.e., 5 to 8 h after the morning meal), whereas the prandial period was defined as the period from 30 min before to 60 min after the morning meal (given at 0900). Digestive enzyme activities and antibacterial activity were determined. Pancreatic protein secretion was 44% less (P < 0.05) in restricted piglets than in control piglets during the basal period. Trypsin secretion was affected by feed-restriction of piglets. The meal did not affect protein and trypsin secretions in restricted piglets, whereas at d 3 postweaning, protein and trypsin secretions and trypsin specific activity in control piglets were 9-, 105-, and 25-fold greater (P < 0.001) during the first 30 min after the meal than before the meal. Lipase and amylase secretions were not affected by variations in feed intake. The secretion of antibacterial activity in restricted piglets was greater (P < 0.05) than that of control piglets only at d 5. The extended feed restriction period increased the basal secretion of antibacterial activity (P = 0.09) and postprandial secretion of amylase (P = 0.05). In conclusion, a low level of feed intake during the early postweaning period decreased pancreatic protein and trypsin secretions, whereas a 2-d feed restriction period enhanced secretions of amylase and antibacterial activity. In addition, our results indicate that during periods of dietary adaptation, such as at weaning, measurements of enzyme activities in the tissue do not accurately reflect the enzyme secretion. 相似文献
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K G Gillette 《Avian diseases》1981,25(2):431-443
Specific-pathogen-free chickens were tested for local antibody response after respiratory exposure to live avian infectious bronchitis virus (IBV). Tracheobronchial secretions were obtained after intraocular-intratracheal vaccinations with Holland and Massachusetts 41 strains IBV. Low levels of immunoglobulin (Ig)A and IgG virus-neutralizing antibodies were detected in secretions after each of two vaccinations with a moderate dose (10(4) EID50), neutralizing activity in secretions was found mainly in IgG antibody. Only challenge exposure to Holland IBV resulted in a marked secondary secretion antibody response. 相似文献
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伪中间葡萄球菌（Staphylococcus pseudintermedius）是犬脓皮病主要致病菌,具有多重耐药性,严重威胁犬及人类健康。本研究从脓皮病患犬中分离20株伪中间葡萄球菌,并揭示大黄酸对伪中间葡萄球菌的抗菌活性及抗菌机理。通过测定最小抑菌浓度、最低杀菌浓度、抑菌曲线和黏附抑制试验分析了大黄酸对伪中间葡萄球菌的抑菌活性;检测大黄酸对伪中间葡萄球菌细胞膜完整性、通透性,细胞活性氧（ROS）水平等,结合电镜观察细菌细胞形态和超微结构,从而探究大黄酸的抑菌机理。结果表明,大黄酸对伪中间葡萄球菌的最小抑菌浓度为12.5 μg·mL-1,亚抑菌浓度能够显著抑制伪中间葡萄球菌生长和黏附;经大黄酸处理后,伪中间葡萄球菌细胞膜通透性改变,胞外β-半乳糖苷酶活性增加（P<0.05）;ROS测定结果显示,大黄酸处理后细菌细胞ROS水平增高3.9倍（1×MIC）和6.4倍（2×MIC）;大黄酸处理后,电镜观察伪中间葡萄球菌表面存在大量分泌物、皱缩,胞壁破裂、电子密度降低、内容物泄漏等。综上,大黄酸可通过改变细菌细胞膜通透性、破坏膜完整性,诱导细菌细胞产生大量ROS等作用机制达到抗菌目的。 相似文献
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OBJECTIVE: To examine the effects of DNA dose, site of vaccination, and coadministration of a cytokine DNA adjuvant on efficacy of H1-subtype swine influenza virus hemagglutinin (HA) DNA vaccination of pigs. ANIMALS: 24 eight-week-old mixed-breed pigs. PROCEDURE: 2 doses of DNA were administered 27 days apart by use of a particle-mediated delivery system (gene gun). Different doses of HA DNA and different sites of DNA administration (skin, tongue) were studied, as was coadministration of porcine interleukin-6 (pIL-6) DNA as an adjuvant. Concentrations of virus-specific serum and nasal mucosal antibodies were measured throughout the experiment, and protective immunity was assessed after intranasal challenge with homologous H1N1 swine influenza virus. RESULTS: Increasing the dose of HA DNA, but not coadministration of pIL6 DNA, significantly enhanced virus-specific serum antibody responses. Pigs that received DNA on the ventral surface of the tongue stopped shedding virus 1 day sooner than pigs vaccinated in the skin of the ventral portion of the abdomen, but none of the vaccinated pigs developed detectable virus-specific antibodies in nasal secretions prior to challenge, nor were they protected from challenge exposure. Vaccinated pigs developed high virus-specific antibody concentrations after exposure to the challenge virus. CONCLUSIONS AND CLINICAL RELEVANCE: Co-administration of pIL-6 DNA did not significantly enhance immune responses to HA DNA vaccination or protection from challenge exposure. However, HA DNA vaccination of pigs, with or without coadministration of pIL-6 DNA, induced strong priming of the humoral immune system. 相似文献
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Lactoferrin (LF), a glycogen of the transferrin family with anti-bacterial and immunomodulatory properties, is expressed in various secretions and tissues. Cutaneous LF serves as a mast cell stabilising compound, modulates T cell activity and is found during IgE-mediated late phase reactions at allergen challenged sites. Culicoides hypersensitivity (CHS) in horses is a common IgE-mediated allergic dermatitis, characterised by an early and late phase cutaneous reaction upon allergen challenge. The aim of the study presented here was to examine whether LF mRNA expression in skin biopsies from horses affected by CHS prior to and 4h following intradermal challenge with a commercial C. nubeculosus extract is modified in comparison to skin biopsies from non-affected horses. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. In comparison to non-affected horses, higher variation in LF mRNA levels both prior to and post-intradermal challenge with C. nubeculosus extract was seen in horses affected by CHS. However, the statistical analysis demonstrated that LF mRNA expression was not significantly different between CHS affected and non-affected horses prior to intradermal challenge with C. nubeculosus extract. Intradermal injection of C. nubeculosus extract did not result in local upregulation of LF mRNA at 4h post-injection. LF mRNA expression was therefore not significantly different pre- or post-intradermal challenge with C. nubeculosus extract in either group. Our data indicate that clinically normal skin of horses affected by CHS is not characterized by modified maintenance levels of LF mRNA. In contrast to human skin allergen challenged sites, LF mRNA levels in horses affected by CHS are not significantly different to that of control sites at 4h post-injection of C. nubeculosus extract. 相似文献
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Serial changes in the concentrations of IgM, IgG and IgA were compared in specific pathogen free (SPF) lambs which had been vaccinated with live or inactivated parainfluenza 3 virus (PI 3) by either intramuscular (IM) or intranasal (IN) routes followed by aerosol challenge with PI 3. In the serum, an increase in IgM was associated with the primary antibody response to the aerosol challenge, whereas increased IgG was associated with the secondary antibody response. No changes in immunoglobulin concentrations were observed in the nasal secretions of lambs administered live or inactivated virus IM or IN without adjuvant. Marked increases in IgG were found in the serum and nasal secretions of lambs vaccinated IM with inactivated virus in Freund's complete adjuvant (FCA) and fractionation by gel filtration confirmed that the antibody was associated with IgG in both these fluids. 相似文献
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Prophylactic efficacy of 4 antibacterial shampoos against Staphylococcus intermedius in dogs was determined by use of a controlled quantitative technique. Ten adult Beagles were used in the study. The antibacterial agents in the shampoos were 3.0% benzoyl peroxide, 0.5% chlorhexidine acetate, 1.0% available iodine as a polyalkyleneglycol-iodine complex, and a combination of 0.5% triclosan, 2.0% sulfur, and 2.0% salicylic acid. Treated and control sites were challenge exposed with 5.30 +/- 0.10 (log10) S intermedius colony-forming units (CFU)/cm2 of skin and occluded for 5 hours. At the end of the test period, remaining bacteria were removed with a detergent cup-scrub technique and the total number of S intermedius CFU/cm2 skin was calculated for each treated and control site. Nontreated bacteria-challenged control sites yielded 5.62 +/- 0.65 S intermedius CFU/cm2 of skin. Staphylococcus intermedius recovery (CFU/cm2) from the treated sites was 0.94 +/- 0.76 for benzoyl peroxide, 1.96 +/- 1.33 for chlorhexidine acetate, 3.11 +/- 0.48 for organic iodine, and 4.69 +/- 0.23 for triclosan-sulfur-salicylic acid. Each S intermedius recovery value from the 4 treated sites was significantly (P less than 0.05) lower than that from the nontreated S intermedius challenge-exposed control site. Bacteria recovery values were also significantly (P less than 0.05) different among the 4 shampoo-treated sites. We concluded that all shampoos had significant (P less than 0.05) prophylactic activity against S intermedius over 5 hours. The shampoo containing benzoyl peroxide was determined to have the greatest efficacy among the products tested. 相似文献
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Anticytotoxin activity of bovine sera and body fluids against Pasteurella haemolytica A1 cytotoxin. 总被引：1，自引：1，他引：0 
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下载免费PDF全文 H J Cho J G Bohac W D Yates H Bielefeldt Ohmann 《Canadian journal of veterinary research》1984,48(2):151-155
Toxin neutralizing activity of bovine sera and body fluids against Pasteurella haemolytica type A1 cytotoxin was evaluated by 51Cr release assay using bovine peripheral blood mononuclear leukocytes as the target cells. Sera collected from precolostral calves did not exert anticytotoxin activity at 10(-1) or higher dilutions, whereas randomly selected complement fixing antibody-negative sera neutralized on average over 90% of cytotoxin activity at the 10(-1) dilution and less than 50% of the toxin activity at 10(-2) or higher serum dilutions. Nasal secretions and lung washings of some of the cattle tested also contained cytotoxin neutralizing activity. The antibody nature of the cytotoxin neutralizing activity was demonstrated by its neutralization with bovine immunoglobulin G2 purified from pooled seropositive sera. Sera from a group of cattle which were vaccinated with a potassium thiocyanate extract of P. haemolytica, but which subsequently developed fibrinous pneumonia after aerosol challenge with bovine herpesvirus 1 and P. haemolytica, had significantly lower anticytotoxin activity than sera from another group of cattle which did not develop the disease after similar vaccination and challenge. Cattle which survived a natural outbreak of shipping fever had higher anticytotoxin activity than those having fibrinous pneumonia in the aforementioned experimental group, although there was no statistical difference between them and a randomly selected CF seronegative group. It is probable that this cytotoxin neutralizing antibody exerts a beneficial effect in protection of cattle against pneumonic pasteurellosis. 相似文献
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Pluta K Irwin JA Dolphin C Richardson L Fitzpatrick E Gallagher ME Reid CJ Crowe MA Roche JF Lonergan P Carrington SD Evans AC 《Journal of animal science》2011,89(12):4032-4042
The cervix and its secretions undergo biochemical and physical changes under the differential influences of estrogen and progesterone. These include changes in the glycoprotein profile of the endocervix and its secretions. A comprehensive survey of such changes in cervical epithelium and cervical secretions was performed on bovine samples throughout the periestrous period. Cervical tissue samples and swabs were collected from synchronized beef heifers that were slaughtered 1) 12 h after controlled intravaginal progesterone-releasing device (CIDR) removal, 2) 24 h after CIDR removal, 3) at the onset of estrus, 4) 12 h after the onset of estrus, 5) 48 h after the onset of estrus, and 6) 7 d after the onset of estrus. Histological staining with hematoxylin and eosin, periodic acid Schiff, Alcian blue, and high-iron diamine was carried out to map overall patterns of stored glycoproteins and tissue structure. Biotinylated lectins were also used to detect the presence and distribution of a range of saccharide structures. The activities of β-galactosidase, α-L-fucosidase, β-N-acetyl-hexosaminidase, and sialidase were measured in cervical swabs using specific substrates. The epithelial layer of the cervix exhibited dynamic changes in cellular hypertrophy and amounts of stored glycoprotein. The greatest content of neutral and acidic mucins was observed 48 h after onset of estrus (P < 0.05). Sialylated mucins predominated at the bases of cervical folds, whereas sulfated mucins were more abundant (P < 0.05) at their apices. The stained area of core mucin glycans changed (P < 0.05) in association with follicular versus luteal phases, whereas terminal glycans changed (P < 0.05) mainly at the time of estrus and shortly thereafter. The greatest activity of β-galactosidase and sialidase was observed 12 h after onset of estrus, whereas β-hexosaminidase and α-fucosidase peaked at the luteal time point (P < 0.05). Taken together, we suggest that the well-known changes in the endocervix and its secretions that are associated with the physiological modulation of sperm transport and function of the cervical barrier are, in part, driven by glycosylation changes. 相似文献