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利用凝胶色谱法对东北林蛙皮肤分泌物中蛋白质及多肽组分分子量的分布进行分析。应用葡聚糖凝胶Sephadex G-100、G-50、G-25,利用已知分子量的牛血清蛋白（Mr 66000）、卵清蛋白（Mr 45000）、细胞色素C（Mr 11700）、胰岛素（Mr 5700）、胰高血糖素（Mr 3550）等作为标准品,对流动相、洗脱速度、色谱条件等进行反复试验,用0．5％甲醇洗脱液（含0．14mol／L NaCl）、0．4ml／min流速,筛选东北林蛙皮肤分泌物中分子量Mr 4000左右的蛋白质及多肽,分离获得了大量相应分子量范围的蛋白及多肽组分。该实验方法简便,结果重现性好,适合作为生物工程产品的质控方法。 相似文献
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<正>抗菌肽作为一种高效的防治药物和天然生物制剂,具有抗菌消炎、抗病毒、免疫调节等多种生物学功能,可以代替抗生素应用在生猪生产中,减少肉制品药物残留。抗菌肽是具有生物活性的小分子多肽。1980年瑞典科学家G.Boman等人经注射阴沟肠杆菌及大肠杆菌诱导惜古比天蚕蛹产生出具有抗菌活性的多肽,定名为Cecropins。抗菌肽分子量在2 000～7 000左右,由20～60个氨基酸残基组成。这类活性多肽具有如热稳定性以及广谱抗菌等特点。 相似文献
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为探讨嗜驼璃眼蜱血淋巴中抗菌肽的电泳分析方法及其抗菌作用，从半饱血的嗜驼璃眼蜱雌蜱体内抽取血淋巴，用50mL／L的乙酸抽提，经AU—PAGE分析嗜驼璃眼蜱血淋巴中阳离子多肽成分，用电泳凝胶琼脂糖弥散法对各多肽进行抗菌活性的筛选。结果表明，嗜驼璃眼蜱血淋巴中阳离子多肽成分对大肠埃希菌和金黄色葡萄球菌均有很强的抗菌作用。AU—PAGE发现一电泳迁徙率明显高于溶菌酶的阳离子抗菌多肽。提示在嗜驼璃眼蜱的血淋巴中，存在与溶菌酶不同的抗菌多肽，可能是嗜驼璃眼蜱抵御感染的重要分子。AU—PAGE是一种较简单、快速、经济的分离纯化抗菌肽的方法。 相似文献
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产肠毒素性大肠杆菌工程菌株pSLM004在限定性培养基上培养后,上清液经硫酸铵沉淀、丙酮抽提、Sephadex G-25凝胶过滤、DEAE-Sephadex A-25离子交换层析、Bio-gel P-4凝胶及反相高压液相层析,获得的STa纯化倍数为302.7倍,有效剂量达10.72ng/鼠单位,回收率为9.9%。纯化的STa在Sephadex G-25凝胶过滤时出现1个蛋白峰,聚丙烯酰胺凝胶电泳(PAGE)呈现1条蛋白带;在100℃30min不失活,121℃15min则失活;在pH 1～10范围内保持活性;胰蛋白酶、链霉蛋白酶对其活性无影响;2-巯基乙醇、二硫苏糖醇(DTT)可使其迅速失活,表明在STa分子中有生物活性所必需的二硫键存在。 相似文献
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目的:建立一种从两栖动物皮肤组织中提取高质量总RNA的方法。方法:改良传统TRIzol法,提取皮肤总RNA,紫外分光光度计测定RNA纯度和得率,1.1%琼脂糖凝胶电泳检测其完整性。结果:改良方法提取的总RNA,其A260/A280值在1.8～2.1之间,A260/A230大于2;琼脂糖凝胶电泳显示清晰的28s rRNA和18s rRNA条带,且28s rRNA条带亮度约为18s rRNA的2倍。结论:改良TRIzol法提取的总RNA纯度高、完整性好、杂质少、得率大,易于操作掌握,可以用于相应的分子生物学试验。 相似文献
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毛瓣棘豆中苦马豆素的纯化与鉴定 总被引：4，自引：1，他引：4
毛瓣棘豆草粉(3.28 kg)经95%乙醇加热回流提取,回收溶剂,浓缩得浸膏。浸膏用蒸馏水混悬,依次用石油醚、氯仿、乙酸乙酯、正丁醇萃取;剩余水溶液用HC l酸化后经氯仿萃取,酸水液用NaOH调pH 12~14得碱水液。碱水液依次用氯仿、乙酸乙酯、正丁醇萃取,得到碱性正丁醇萃取物5.7 g。碱性正丁醇萃取物经硅胶柱层析,用氯仿-甲醇梯度洗脱,TLC(薄层色谱)检测,合并相同组份,其中101~200份含有苦马豆素。将101~141份用氨性氯仿处理,减压升华,得到白色针状结晶34 mg。经TLC(薄层色谱)检查、熔点测定和质谱分析可确定该白色针状结晶为苦马豆素。 相似文献
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为了建立大量获取中国林蛙抗菌肽的方法,根据GenBank中的中国林蛙皮肤抗菌肽Temporin-1CEa基因的mRNA序列(EU624139)设计一对特异性引物,以提取的中国林蛙皮肤总RNA反转录出的cDNA为模板,将扩增的编码序列与pEASY-T3克隆载体连接获得Tem-T3;利用酶切、连接等分子生物学手段,将GFP基因连入Tem-T3克隆载体,再经酶切获得Tem-GFP片段,并插入真核表达载体pcDNA3.1,最终得到Tem-GFP-pcD-NA3.1重组质粒;利用脂质体转染法将该质粒转入绵羊成纤维细胞,48 h后可在荧光倒置显微镜下观察到GFP的绿色荧光表达;qPCR数据分析显示,与对照组相比,转染Tem-GFP-pcDNA3.1的绵羊成纤维细胞中融合蛋白Tem-GFP的表达量可提高约300倍。本研究为构建Temporin-1CEa基因山羊乳腺特异表达载体提供依据。 相似文献
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An in situ system involving incubation of 60- to 80-g pieces of muscle at 4 degrees C under different conditions was used to determine the effects of time of postmortem storage, of pH, and of temperature on activities of mu- and m-calpain activity in bovine skeletal muscle. Casein zymograms were used to allow measurement of calpain activity with a minimum of sample preparation and to ensure that the calpains were not exposed to ionic strengths of 100 or greater before assay of their activities. In 4 of the 5 muscles (longissimus dorsi, lumbar; longissimus dorsi, thoracic; psoas major; semimembranosus; and triceps brachii) studied, mu-calpain activity decreased nearly to zero within 48 h postmortem. Activity of m-calpain also decreased in the in situ system used but at a much slower rate. Activities of both mu- and m-calpain decreased more slowly in the triceps brachii muscle than in the other 4 muscles during postmortem storage. Although previous studies have indicated that mu-calpain but not m-calpain is proteolytically active at pH 5.8, these studies have used calpains obtained from muscle at death. Both mu- and m-calpain are proteolytically inactive if their activities are measured at pH 5.8 and after incubating the muscle pieces for 24 h at pH 5.8. Western analysis suggested that neither the large 80-kDa subunit nor the small 28-kDa subunit of m-calpain was autolyzed during postmortem storage of the muscle pieces. As has been reported previously, the 80-kDa subunit of mu-calpain was autolyzed to 78- and then to a 76-kDa polypeptide after 7 d postmortem, but the 28-kDa small subunit was not autolyzed; hence, the autolyzed mu-calpain molecule in postmortem muscle is a 76-/28-kDa molecule and not a 76-/18-kDa molecule as previously assumed. Because both subunits were present in the postmortem calpains, loss of mu-calpain activity during postmortem storage is not due to dissociation of the 2 subunits and inactivation. Although previous studies have shown that the 76-/18-kDa mu-calpain molecule is completely active proteolytically, it is possible that the 76-/28-kDa mu-calpain molecule in postmortem muscle is proteolytically inactive and that this accounts for the loss of mu-calpain activity during postmortem storage. Because neither mu- nor m-calpain is proteolytically active at pH 5.8 after being incubated at pH 5.8 for 24 h, other proteolytic systems such as the caspases may contribute to postmortem proteolysis in addition to the calpains. 相似文献
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MAO Xin ZHANG Guizhen QU Jinyao ZHANG Yuhao LU Guozhu GAO Yonglin LI Yanshen 《中国畜牧兽医》2007,47(12):4093-4102
The aim of this study was to search for novel non-ribosomal peptide antimicrobial substances based on the screening of bacterial secondary metabolites.The bacteria isolated from soil,sea water and common marine organisms in Yantai coastal area were isolated and purified.E.coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were selected as indicator bacteria,and E.coli B2 (blaNDM-5+mcr-1) and methicillin-resistant Staphylococcus aureus (MRSA) T144 were used as indicator for secondary screening.The genome was extracted and the PCR products were sequenced to determine the active species.The secondary metabolites of bacteria were extracted by organic extraction,purified by gel chromatography and preparative liquid chromatography,and purity was detected by analytical liquid chromatography.The results of sequencing showed that the active strain belonged to Bacillus amyloliquefaciens sp.,and was named as Bacillus amyloliquefaciens 9-14 (active bacterium 9-14).The results of antibacterial test showed that the metabolites of active bacterium 9-14 had high inhibitory effect on Staphylococcus aureus ATCC 29213,MRSA T144,E.coli ATCC 25922 and E.coli B2.The metabolites of active bacterium 9-14 were cyclic lipopeptides composed of amino acid chains,which belonged to the derivatives of ibuprofen.The biological characteristics and antibacterial spectrum of the metabolites of active bacterium 9-14 were studied.The results showed that the metabolites of active bacterium 9-14 had good thermal stability and acid-base stability.And the antibacterial activity of the antibacterial substance treated with trypsin,pepsin,protease K and papain was not significantly weakened and had good stability.The antibacterial substance also had inhibitory effect on Staphylococcus aureus and E.coli,but had no activity against Pseudomonas aeruginosa,Klebsiella pneumoniae,Enterococcus faecalis and Bacillus cereus.In this study,a new antibacterial substance was obtained,which could be used as the precursor of antibacterial drugs,and could provide certain reference for food safety and disease control. 相似文献
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本研究旨在基于对细菌次级代谢产物的筛选,寻找新型非核糖体肽类抗菌物质。通过对烟台沿海地区的土壤、海水及近海常见海洋生物中的细菌进行培养,分离纯化得到细菌的单克隆,以大肠杆菌（E.coli）ATCC 25922和金黄色葡萄球菌ATCC 29213为指示菌进行初步筛选,以耐多黏菌素和碳青霉烯E.coli B2（blaNDM-5+mcr-1）和耐甲氧西林金黄色葡萄球菌（MRSA） T144作为指示菌对有活性的细菌进行二次筛选。通过提取基因组进行PCR扩增产物测序比对,确定活性菌种属。采用有机萃取法对细菌的次级代谢产物进行萃取,通过凝胶层析和制备液相色谱进行纯化,利用分析型液相色谱进行纯度检测,进—步利用质谱对纯化后的抗菌活性物质进行结构鉴定。测序结果表明,活性菌属于解淀粉酶芽孢杆菌种,将其命名为解淀粉酶芽孢杆菌9-14（活性菌9-14）。抑菌试验结果表明,活性菌9-14的代谢产物对金黄色葡萄球菌ATCC 29213、MRSA T144、E.coli ATCC 25922和E.coli B2均具有高效抑制作用。活性菌9-14代谢产物是由氨基酸链组成的环状脂肽,属于伊枯草菌素的衍生物。对活性菌9-14代谢产物的生物学特性及其抑菌谱研究发现,活性菌9-14的代谢产物具有良好的热稳定性和酸碱稳定性,该抗菌物质经胰蛋白酶、胃蛋白酶、蛋白酶K和木瓜蛋白酶处理后抗菌活性没有明显减弱,具有较好的稳定性;该抗菌物质对所用金黄色葡萄球菌和大肠杆菌同样具有抑制作用,对绿脓杆菌、肺炎克雷伯杆菌、粪肠球菌和蜡样芽孢杆菌均不表现活性。本研究得到一种新型的抗菌物质,以该抗菌物质为抗菌药物的前体,可为食品安全和疾病控制提供一定的参考依据。 相似文献
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华南虎幼崽为晚成哺乳动物,刚出生时双眼未睁、不能独立取食和排便,需要人工辅助其学习和完成。初生仔兽的喂饲以少量多食为佳,前3 d以7～8次较为适宜,喂奶量由少增多。饲喂幼虎时人工奶的初始浓度以1：8较适合。幼兽需要用纱布蘸水轻擦仔兽的肛周,以刺激其排便。在8周龄左右为幼虎添加肉浆,1个月后将肉浆替换为肉条。肉类食物的添加时间为观察到幼虎牙齿长出,此时喂奶量可以随着肉量的增加而逐渐减少,直至完全断奶。饲料转换过程中,动物对人工奶的取食量逐渐下降,并且在1周内会出现身体不适,体重增长减缓,一般持续5～7 d即恢复正常。动物对肉条适应以后,而对人工奶的需求量急剧下降,此时可以增加骨头以锻炼幼兽的啃食能力,此时幼兽的体重迅速上升。 相似文献