稳定表达禽β防御素6的DF-1细胞系的建立及其抑菌活性

[背景]禽β防御素6是禽体内分泌的一类抗菌肽,在抵抗病原入侵和免疫调节中发挥着重要作用,但其常规表达方式效率较低,难以在产业化生产中加以应用.[目的]建立稳定表达AvBD6的细胞系,并检测其表达产物对耐药大肠杆菌的抗菌活性,为其他防御素表达提供参考.[方法]利用显微镜观察构建真核重组表达载体pLOV-eGFP-AvBD...     

小菜蛾溶菌酶Pxlys的基因克隆及其重组蛋白的抗菌活性分析

[目的]本研究旨在通过研究小菜蛾Plutella xylostella溶菌酶的功能,进一步认识小菜蛾的免疫防御机理,为小菜蛾的生物防治提供新的思路.[方法]利用RACE技术克隆小菜蛾溶菌酶基因.构建原核表达载体pET-29a-Pxlys,利用原核表达系统表达并用镍柱亲和层析纯化重组蛋白Pxlys.利用牛津杯法检测重组蛋...     

Induction, selection and antibacterial activity of the antibacterial peptides from lepldopteran insect cultured cell lines

We induced 3 cell lines that were in vitro cultured from Lepidoptera with heat inactivated Escherichia coil DH5α to stimulate the antibacterial peptide followed by antibacterial activity assay,induction dynamic research and Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) experiment.The antibacterial activity of the induced BTI-Tn-5B1 cell line was the highest,and the antibacterial activity increased gradually to the highest level in 16 hours after stimulation.A new antibacterial peptide with a molecular weight of about 8000 Da was preferentially induced in Trichoplusia ni BTI-Tn-5B1 ceils in 16 hours after stimulation.Antibacterial activity assays indicated that it had inhibition against Staphylococcus aureus,Escherichia coli K12D31 and Salmonella derby.It has especially strong inhibition against Gram-negative bacteria such as Escherichia coli KI2D31 and Salmonella derby.     

重组毕赤酵母产青蛤Mytimacin抗菌肽的表达研究

Mytimacin是主要在无脊椎动物中表达的Macin抗菌肽家族中的一员,具有较强的抗病原微生物活性,是利用重组DNA技术开发天然抗菌剂的良好候选者。通过RT-PCR从青蛤(Cyclina sinensis)闭壳肌中克隆编码Mytimacin成熟肽的基因,经3次PCR在该基因的5’端添加Xho I限制性酶切位点和信号肽酶识别位点、3’端添加Xba I限制性酶切位点和6×His,获得目的基因"CsMm";以pPICZαA为表达载体、毕赤酵母(Pichia pastoris)X-33为工程菌,构建重组毕赤酵母X-33/pPICZαA-CsMm。通过高浓度博来霉素筛选高拷贝酵母转化子,在28℃、250 r/min条件下,使用1.5%的甲醇诱导表达72 h;使用固化金属离子亲和层析(IMAC)对表达产物进行纯化,并通过MALDI-TOF-TOF质谱分析对纯化产物进行鉴定。另外,通过涂布法和浊度法考察重组CsMm的抑菌活性。结果表明:基于X-33/pPICZαA-CsMm重组毕赤酵母的外源表达获得了表达量为25.6 mg/L的重组蛋白,经MALDI-TOF-TOF质谱鉴定其为分子量约7.8 kD的预期重组CsMm。抑菌试验证明重组CsMm对金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、大肠杆菌(Escherichia coli)和副溶血性弧菌(Vibrio Parahemolyticus)具有明显的抑菌活性。构建的重组毕赤酵母X-33/pPICZαA-CsMm能有效合成具有生物学活性的重组青蛤Mytimacin,旨为贝类来源天然小分子抗菌剂的开发提供可资参考的技术途径。     

一种抗菌肽和aFGF融合蛋白的构建和表达

利用PCR技术扩增出带有凝血酶Xa因子切割位点的天蚕素蜂毒素杂合肽和aFGF的融合基因,插入大肠杆菌表达载体pET-3c中,构建出表达质粒pET-aF-CM,并转化至大肠杆菌BL21（DE3）中,氨苄青霉素抗性筛选重组转化子。IPTG诱导4h后,以包涵体形式表达的融合蛋白约占菌体总蛋白的17%。将包涵体溶解后透析复性,并利用肝素亲和层析纯化,得到电泳纯的融合蛋白。Western blot分析表明,该蛋白能与aFGF抗体产生免疫反应。MTT法检测显示,融合蛋白具有促3T3Bal/b细胞分裂活性,其比活为1.471×106IU/mg。利用凝血酶Xa因子裂解融合蛋白,可以获得抗菌肽和含凝血酶Xa因子裂解序列的aFGF蛋白。分子筛回收含杂合抗菌肽,抑菌活性检测表明其对大肠杆菌K12D31具有明显抑菌活性。微量稀释法检测结果表明,回收的抗菌肽对大肠杆菌DH5α、大肠杆菌K12D31、沙门氏菌、金黄色葡萄球菌、枯草芽孢杆菌和绿脓杆菌的MIC分别达6.25μg/ml、10μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml和5μg/ml。     

抗菌肽GK1在大肠杆菌中的融合表达

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。     

紫穗槐种子提取物的抑菌活性研究

目的研究紫穗槐种子提取物的抑菌活性。方法将紫穗槐种子乙醇提取物分别通过石油醚、乙酸乙酯和正丁醇萃取,选择金黄色葡萄球菌和肺炎克雷伯杆菌为供试菌,采用试管二倍稀释法测定紫穗槐种子提取物的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）,涂布平板法绘制杀菌曲线,电镜下观察药物对细菌超微结构的影响。结果紫穗槐种子提取物经乙酸乙酯萃取后对供试菌抑制作用较强,其中对金黄色葡萄球菌的MIC和MBC分别为2．5、5．0mg／mL;对肺炎克雷伯杆菌的MIC和MBC分别为5．0、10．0mg／mL;杀菌曲线结果表明,药物对供试菌的抑制作用存在浓度和时间依赖性;电镜结果说明,药物的作用可能与破坏菌体细胞壁、改变细胞膜通透性有关。结论紫穗槐种子提取物具有显著的抗菌活性。     

Synthesis and pharmacological activities of some mononuclear Ru(II) complexes

A series of mononuclear Ru(II) complexes of the type [Ru(M)2(U)]2+, where M = 2,2'-bipyridine/1,10-phenanthroline and U = tpl (Ru1), 4-Cl-tpl (Ru2), 4-CH3-tpl (Ru3), 4-CH3O-tpl (Ru4), and 4-NO2-tpl (Ru5), -pai (Ru6), where tpl = thiopicolinanilide and pai = 2-phenyl-azo-imidazole, have been prepared and characterized by IR, UV-Vis, 1H NMR, 13C-NMR, FAB-Mass spectrophotometer, and elemental analysis. The complexes display metal-ligand charge transfer (MLCT) transitions in the visible region. The title complexes were subjected to in vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's ascitic carcinoma (EAC) and in vitro antibacterial activity against Gram positive and Gram negative microorganisms. Ru1-Ru6 were found to increase the life span of the tumor hosts by 19-52%, and decreased tumor volume and viable ascitic cell count. The results of the present study clearly demonstrated the tumor inhibitory activity of the ruthenium chelates against transplantable murine tumor cell line. The treatment with ruthenium complexes could be secondary to tumor regression or due to the action of the compounds itself. The significant antibacterial activity was observed for Ru1-Ru4 against microorganisms like Vibrio cholera 865, Staphylococcus aureus 6571, and Shigella flexneri as compared to that of standard drug chloramphenical. Ru5 showed moderate activity against S. aureus 8530. However, all the complexes fail to show significant antibacterial activity against V. cholera 14033 and Shigella sonnai.     

Antibacterial activity of honey and propolis from Apis mellifera and Tetragonisca angustula against Staphylococcus aureus

AIMS: The antibacterial activity against Staphylococcus aureus of honey and propolis produced by Apis mellifera and Tetragonisca angustula was evaluated. Secondary aims included the study of the chemical composition of propolis and honey samples and its relationship with antibacterial activity against S. aureus. METHODS AND RESULTS: The antibacterial activity of honey and propolis was determined by the method of macrodilution. The minimum inhibitory concentration (MICs) of A. mellifera honey ranged from 126.23 to 185.70 mg ml(-1) and of T. angustula from 142.87 to 214.33 mg ml(-1). For propolis, the MIC ranged from 0.36 to 3.65 mg ml(-1) (A. mellifera) and from 0.44 to 2.01 mg ml(-1) (T. angustula). Honey and propolis were evaluated by high-performance liquid chromatography. Some typical compounds of Brazilian propolis were also identified in honey samples. Principal component analysis revealed that the chemical composition of honey and propolis samples was distinct based on the geographical location of the samples. CONCLUSIONS: Propolis samples had higher antibacterial activity against S. aureus when compared with honey. However, both propolis and honey samples had antibacterial against S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: These antimicrobial properties would warrant further studies on the clinical applications of propolis and honey against S. aureus.     

In vitro effect of some Indian honeys on Staphylococcus aureus from wounds

Staphylococcus aureus is the most frequently isolated pathogen from wounds with multiple resistances to antibiotics. Honey has been demonstrated and reported to be effective antibacterial agent on Gram positive and Gram negative organisms. Hence, the present study was conducted to evaluate the in vitro antibacterial effect of Indian honeys on Staphylococcus aureus obtained from wounds. A total of 123 Staphylococcus aureus isolates along with ATCC 25923 were categorized as sensitive, multi drug resistant (MDR) and non-MDR strains. Out of total nine Indian honeys (three each of unifloral, multifloral and branded marketed honey) used, three unifloral and three multifloral honey samples showed antibacterial activity against all the organisms tested by Agar diffusion method but not the branded marketed honeys. The MIC values of all honey samples for all studied Staphylococcus aureus isolates ranged between 5-15% (v/v). Unifloral honey samples showed higher antibacterial activity than multifloral honey. The single sample of Jambhul honey showed the highest activity. Thus, Indian honeys were found to be effective for their antimicrobial activity on sensitive, non-MDR, MDR and ATCC strains of S. aureus.     

Antimicrobial activity of endemic Crataegus tanacetifolia (Lam.) Pers and observation of the inhibition effect on bacterial cells

Up to now an increasing number of antibiotic-resistant bacteria have been reported and thus new natural therapeutic agents are needed in order to eradicate these pathogens. Through the discovery of plants such as Crataegus tanacetifolia (Lam.) Pers that have antimicrobial activity, it will be possible to discover new natural drugs serving as chemotherapeutic agents for the treatment of nosocomial pathogens and take these antibiotic-resistant bacteria under control. The objective of the present study was to determine antimicrobial activity and the activity mechanism of C. tanacetifolia plant extract. The leaves of C. tanacetifolia, which is an endemic plant, were extracted using methanol and tested against 10 bacterial and 4 yeast strains by using a drop method. It was observed that the plant extract had antibacterial effects on Bacillus subtilis, Shigella, Staphylococcus aureus, and Listeria monocytogenes among the microorganisms that were tested. Minimum inhibitory concentration (MIC) results obtained at the end of an incubation of 24 h were found to be > or =6.16 mg ml(-1) for B. subtilis, < 394 mg ml(-1) for Shigella, and > or =3.08 mg ml(-1) for L. monocytogenes and S. aureus and minimum bactericidal concentration (MBC) were found as > or =24.63 mg ml(-1) for B. subtilis, > or =394 mg ml(-1) for Shigella, > or =6.16 mg ml(-1) for L. monocytogenes, and > or =98.5 mg ml(-1) for S. aureus. According to the MBC results, it was found that the plant extract had bactericidal effects and in order to explain the activity mechanism and cell deformation of bacterial strains treated with plant extract, the scanning electron microscopy (SEM) was used. The results of SEM showed that the treated cells appeared shrunken and there was degradation of the cell walls. This study, in which the antibacterial effect of C. tanacetifolia was demonstrated, will be a base for further investigations on advanced purification and effect mechanism of action of its active compounds.     

鹅β-防御素3基因的分离、鉴定及其表达产物的生物学特性

为了克隆鹅β-防御素(AvBD)3基因,并在原核表达重组鹅AvBD3蛋白,进一步研究鹅AvBD3蛋白的生物学特性,利用RT-PCR方法从鹅脾脏和法氏囊组织中扩增到鹅AvBD3基因片段,其cDNA片段大小为182 bp,编码60个氨基酸残基.经同源性分析发现鹅AvBD3氨基酸序列与鸡AvBD3氨基酸序列同源性最高,为100％.将该基因亚克隆到原核表达载体pGEX-6p-1的BamH Ⅰ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-goose AvBD3.将重组质粒转化大肠杆菌BL21,于37℃用IPTG诱导表达,SDS-PAGE电泳表明,重组鹅AvBD3蛋白在原核高效表达(分子量约31 kDa).该重组蛋白经纯化后测定其体外抗菌活性与理化特性,结果显示,重组鹅AvBD3蛋白具有广谱的抗菌活性,对12种细菌,包括革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用.高盐离子浓度显著降低重组鹅AvBD3蛋白的抗菌活性.此外,该重组蛋白的溶血活性极低,并对酸碱度具有较高的稳定性.     

鸽β-防御素1基因的克隆及其生物学作用鉴定

【目的】从鸽子组织中克隆鸽子β-防御素1(AvBD1)基因,在大肠杆菌中表达重组鸽子AvBD1蛋白,测定其生物学特性。【方法】应用RT-PCR法从鸽子骨髓组织中扩增鸽子AvBD1基因,采用Real-time PCR法检测该基因在鸽子组织器官中的表达分布。将该基因亚克隆到大肠杆菌原核表达载体pProEX-HTa的EcoR I和Xho I双酶切位点上,构建重组表达质粒pProEX-pigeon AvBD1,将重组质粒进行诱导表达;对该重组蛋白进行纯化,通过菌落计数法测定其体外抗菌活性与理化特性。【结果】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其cDNA大小为198 bp,编码65个氨基酸,经序列相似性分析,鸽子AvBD1与鸭AvBD1氨基酸序列相似性最高(81.5%)。鸽子AvBD1主要分布于免疫系统和消化系统组织中。Tricine-SDS-PAGE电泳结果表明,重组鸽子AvBD1蛋白分子量约8.8 kD,与预期大小一致。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性。此外,该重组蛋白的溶血活性极低。【结论】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其主要分布在机体的免疫系统和消化系统中。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性,且该重组蛋白的溶血活性极低。     

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.     

弱酸性家蝇蛆抗菌肽MD7095的分离纯化及性质研究

家蝇抗菌肽多是碱性蛋白,目前尚无弱酸性家蝇抗菌肽的报道。通过稀醋酸低温浸提,海藻酸吸附,稀盐酸低温洗脱、盐析、Sephadex G25凝胶过滤和CMC23弱阳离子交换柱层析等方法,利用灵敏的杀菌活性检测手段,从家蝇蛆(Musca domesticalarvae)中分离纯化出一组弱酸性抗菌肽,对苏云金芽孢杆菌(Bacillus thuringiensis)等革兰氏阳性菌和几种革兰氏阴性菌有强烈的杀灭作用,有极强的耐热、耐冻融的特性。通过电洗脱方法进一步纯化出抗菌肽MD7095,质谱测定其分子量7095Da,IEF电泳测得其等电点5.59,经肽质量指纹谱(PMF)鉴定为一新肽。扫描电镜超微结构观察表明,弱酸性家蝇蛆抗菌肽对苏云金芽孢杆菌的杀菌机制主要是使细胞膜穿孔,内容物外泄,最终使细菌完全解体死亡。     

Antimicrobial activity of Brazilian copaiba oils obtained from different species of the Copaifera genus

The antimicrobial activity of copaiba oils was tested against Gram-positive and Gram-negative bacteria, yeast, and dermatophytes. Oils obtained from Copaifera martii, Copaifera officinalis, and Copaifera reticulata (collected in the state of Acre) were active against Gram-positive species (Staphylococcus aureus, methicillin-resistant S. aureus, Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus faecalis) with minimum inhibitory concentrations ranging from 31.3-62.5 microg/ml. The oils showed bactericidal activity, decreasing the viability of these Gram-positive bacteria within 3 h. Moderate activity was observed against dermatophyte fungi (Trichophyton rubrum and Microsporum canis). The oils showed no activity against Gram-negative bacteria and yeast. Scannning electron microscopy of S. aureus treated with resin oil from C. martii revealed lysis of the bacteria, causing cellular agglomerates. Transmission electron microscopy revealed disruption and damage to the cell wall, resulting in the release of cytoplasmic compounds, alterations in morphology, and a decrease in cell volume, indicating that copaiba oil may affect the cell wall.     

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b,a new avian defensin from the king penguin,against Salmonella enteritidis CVCC3377

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.     

中国明对虾溶菌酶基因克隆、重组表达与性质分析

溶菌酶是机体先天免疫系统中一个重要的效应分子, 参与机体多种免疫反应, 在溶菌过程中形成一个水解体系, 破坏和消除侵入体内的病原, 从而实现机体的免疫防御。从中国明对虾中克隆得到了溶菌酶基因(称为FcLyz基因), 该基因全长709 bp, 其完整的阅读框为477 bp, 编码158个氨基酸, 前18个氨基酸(-1~-18)为信号肽, 成熟肽由140个氨基酸组成(1-140aa), 其分子量为16.2 kD。经SMART分析,该基因具有1个溶菌酶1(LYZ1)结构域(19-130aa)。半定量RT-PCR分析结果表明溶菌酶虽在多种组织中有较低水平的组成性表达, 但在细菌诱导的血细胞、心脏、肝胰腺和鳃等多种组织中表达上调。将中国明对虾溶菌酶基因的成熟肽亚克隆进原核表达载体pET-30a (+)中, 转化大肠杆菌BL21(DE3), 再进行诱导表达和亲和纯化, 得到了纯化的重组溶菌酶, 并进行了抑菌活性检测。结果表明, 重组对虾溶菌酶对革兰氏阳性菌的抑菌能力较强, 最小抑菌浓度达到3.43 mmol/L, 但对革兰氏阴性菌抑制作用较小。上述结果表明, 该溶菌酶作为一种重要的免疫效应分子, 参与了对虾的免疫防御反应。