鸡β-防御素-3在毕赤酵母中的分泌表达及其生物活性测定

采用RT-PCR方法从鸡外周血淋巴细胞中扩增鸡β-防御素-3成熟肽基因,将其克隆到酵母表达载体pPICZα-A的分泌信号肽基因下游.线性化后的重组质粒电击转化毕赤酵母宿主菌GS115,经抗生素Zeocin筛选和PCR鉴定后,得到重组酵母菌.阳性转化菌经甲醇诱导后,SDS-PAGE和western blot鉴定结果表明,在毕赤酵母中得到分泌性表达产物,分子量约为8.9 ku,活性检测表明其具有显著的抑菌活性.     

粪肠球菌产伏尔加霉素E5的生物学特性研究

为了确定分离自健康猪胃肠道粪肠球菌产生的细菌素伏尔加霉素E5(enterocin E5)的生物学特性,本研究检测了该细菌素对热、酸碱、酶类的耐受性及其抗菌谱,enterocin E5经过121℃高压处理10min对指示菌抑菌活性没有影响;细菌素于pH 2～12作用后,对大肠埃希菌仍有抑菌活性,最强的pH抑菌活性范围为pH 4～6;enterocin E5对胰蛋白酶和蛋白酶K处理敏感,过氧化氢酶、脂肪酶和淀粉酶对其抑菌活性没有影响;enterocin E5对21株动物源病原微生物具有抑制作用,革兰阴性病原菌包括大肠埃希菌、鸡白痢沙门菌、鸡伤寒沙门菌、多杀性巴氏杆菌等,特别对产生多重抗药性的3株鸡伤寒沙门菌具有明显的抑菌活性。抑制革兰阳性金黄色葡萄球菌、马腺疫链球菌、猪链球菌,对炭疽芽胞杆菌弱毒株和蜡样芽胞杆菌产生显著的抑菌活性。结果表明,enterocin E5对酸碱是比较稳定的,具有很宽泛的pH耐受范围,属于热稳定IIa亚型细菌素,能够作为一种抗菌物质防控动物源细菌病。     

伴大豆球蛋白酶解肽抑菌活性研究

建立伴大豆球蛋白酶解产生抑菌肽的最适条件,对酶解产物进行分离纯化后进一步测定其抑菌活性。采用复合风味蛋白酶水解伴大豆球蛋白,测定不同时间的酶解液对大肠杆菌和金黄色葡萄球菌的抑菌活性。结果显示:2 h水解产生的酶解肽具有抑菌活性,其对大肠杆菌和金黄色葡萄球菌的最小抑菌浓度分别是2.6和3.2 mg/mL。2 h水解液经Sephadex-G15分离纯化,采用平板计数法鉴定各组分。结果显示:CP2组分对大肠杆菌具有显著的抑制作用(P<0.05)。本研究证明伴大豆球蛋白的复合风味蛋白酶酶解肽具有一定的抑菌活性。     

Gal-13成熟肽基因在毕赤酵母中的表达与抑菌活性分析

扩增鸡β-防御素-13(Gal-13)成熟肽基因序列,构建酵母重组表达载体pPICZαA-Gal-13,通过SacⅠ限制性内切酶线性化处理后,电击转化毕赤酵母X-33,并对利用甲醇诱导后的重组菌株表达产物进行表达规律分析和抑菌活性研究。结果表明,扩增到的Gal-13成熟肽基因序列为108bp,共编码36个氨基酸;筛选到的重组菌株均为Mut+表型,其诱导表达上清经Tricine-SDS-PAGE凝胶电泳后,发现其在约5 900的位置出现目的条带,抑菌试验发现其对沙门菌(CVCC534)和金黄色葡萄球菌(ATCC25923)具有抑菌活性。     

比较益生菌和中药组合对鸡源大肠杆菌抑菌效果分析

试验比较多种益生菌和中药组合对鸡源大肠杆菌的抑菌效果,并对其抑菌机理进行分析。结果表明:益生菌中,枯草芽孢杆菌和植物乳杆菌对鸡源大肠杆菌具有明显的抑菌活性,但地衣芽孢杆菌没有效果。中药组合中,黄连和茯苓、赤芍、金银花、鱼腥草、桂枝分别配伍时抑菌效果明显,但中药组合的抑菌效果明显低于益生菌。进一步研究益生菌中的抑菌成分,对植物乳杆菌和枯草芽孢杆菌发酵液进行粗提取、分离和纯化,发现地衣芽孢杆菌和枯草芽孢杆菌可以产生肠杆菌肽,肠杆菌肽是抗菌肽的一种,其对鸡源大肠杆菌有抑制作用。     

饲用微生态制剂益生特性的研究

研究采用平板扩散抑菌试验和体外细胞黏附模型探究某种饲用微生态制剂的抑菌效果和鸡肠上皮细胞黏附能力。试验结果表明:此种微生态制剂抑菌活性随着质量浓度增大而增强,抑菌的最适pH约为5.0,最适温度约为37℃,具有一定的耐酸性及耐热性;每100个鸡肠上皮细胞上黏附数平均为(3 044.38±58.21)个,黏附率平均为(20.39±0.39)%。     

乳酸菌体外抑菌试验报告

对乳酸杆菌(LL)、嗜酸杆菌(LA)、乳酸链球菌(SL)的抑菌特性进行了初步探讨。采用牛津杯法对3株乳酸菌菌液、菌体及其代谢产物进行体外抑菌试验。结果显示,3株乳酸菌的菌液及其代谢产物具有较强的抑菌活性,而菌体没有抑菌活性。代谢产物热稳定性较好,但受pH影响较大。随着pH升高,抑菌活性逐渐降低,在弱酸性、中性和碱性条件下没有抑菌活性。代谢产物的抑菌活性不受过氧化氢酶、胃蛋白酶、胰蛋白酶的影响。     

枯草芽孢杆菌J-4菌株胞外产抑菌物质性质的初探

为了研究枯草芽孢杆菌(Bacillus subtilis)J-4菌株胞外产抑菌物质的性质,试验采用琼脂打孔扩散法,以大肠杆菌(Escherichia coli)为指示菌,考察硫酸铵盐析、透析处理、水浴处理、缓冲液pH值、蛋白酶、变性剂、离子种类等对该抑菌物质活性的影响。结果表明:J-4菌株胞外产生的抑菌物质可被80%饱和度的硫酸铵盐析;该抑菌物质的分子质量在8.0 ku以上;100℃水浴60 min,该抑菌物质仍有较高抑菌活性;在pH值为2.0~8.0范围内均有较高活性;抑菌物质分子结构中含有可被胃蛋白酶或胰蛋白酶酶解的肽键;抑菌物质的活性可被十六烷基三甲基溴化铵(CTAB)和高浓度的CaCl_2增强。说明枯草芽孢杆菌J-4菌株胞外产生的抑菌物质为抗菌蛋白类物质,具有较高的抗菌活性,热稳定性好,耐受鸡消化道pH值为4.0~7.0环境和消化酶,在养鸡业极具应用潜力。     

酸马奶提取植物乳杆菌DSM20174细菌素的理化特性研究

为研究从酸马奶中提取的植物乳杆菌DSM20174细菌素的理化特性,本试验提纯植物乳杆菌DSM20174细菌素,经过硫酸铵沉淀、透析后得到细菌素粗提液,再利用SephadexG-100凝胶柱层析进行纯化后,细菌素的比活力明显增加,达到154.70 AU/mL,得率为43.5%.采用牛津杯法测定纯化的植物乳杆菌DSM20174细菌素在3种不同方式处理下对牛源野生致病性大肠杆菌O78抑菌效果的影响.结果显示:①植物乳杆菌DSM20174细菌素在5组温度处理下,随着温度的升高抑菌作用降低,各组抑菌作用差异显著(P<0.05).即使121 ℃处理30 min后,抑菌直径仍达14.90 mm.②植物乳杆菌DSM20174细菌素在pH 2.0~12.0由低到高的11组酸碱处理下,pH越大抑菌直径越小,且除了pH 9.0和pH 10.0之间差异不显著(P>0.05)外,其余各组抑菌作用均差异显著(P<0.05). ③植物乳杆菌DSM20174细菌素经胰蛋白酶、胃蛋白酶、木瓜蛋白酶3种酶处理后抑菌直径变化明显,处理前后抑菌作用均差异显著(P<0.05).结果表明植物乳杆菌DSM20174细菌素是较好的抑菌活性物质,但不是热稳定性物质;具有较广泛的pH适用范围,且pH越小时其抑菌活性越强,在最适生长pH时抑菌活性有所增强;不是蛋白酶稳定性物质.     

蜂王浆抗革兰氏阳性菌的特性研究

以枯草芽孢杆菌、金黄色葡萄球菌、四联球菌作为对象菌,利用抑菌圈试验法研究了蜂王浆抗革兰氏阳性菌的特性。实验结果表明：蜂王浆对3种供试革兰氏阳性菌都具有抑制作用,其抑菌活性随浓度的增加而增加;在所试验的pH值范围内（3．0-9．0）,蜂王浆对枯草芽孢杆菌和金黄色葡萄都表现出了抑制作用,而只有当pH值分别为9,4,3时才对四联球菌表现出抑制作用。蜂王浆的抑菌活性随贮存温度和贮存时间的变化而变化,在-20℃～25℃时贮藏15d后,蜂王浆对枯草芽孢杆菌的抑菌性能将会明显下降;在-20℃贮藏25d后以及在5℃、15℃、25℃贮藏15d后,蜂王浆对金黄色葡萄球菌的抑菌活性会显著下降;蜂王浆对于四联球菌的抑制作用似乎更稳定,在所试验的温度范围及时间范围内,其抑菌活性未出现显著下降。蜂王浆的抗菌成分主要是醚溶物和水溶物,而醚水不溶物不具有抑菌活性。该结果对蜂王浆抗菌物质的进一步开发利用提供了前期基础。     

多黏类芽孢杆菌抗菌肽的分离纯化及特性研究

试验旨在分离纯化多黏类芽孢杆菌（Paenibacillus polymyxa）BLCC1-0402代谢产物中的抗菌肽,为抗菌肽制备及其制品检测提供参考。采用离心、不同分子质量卷式膜超滤浓缩、Superdex peptide 10/300GL凝胶过滤层析对多黏类芽孢杆菌发酵上清液进行逐级分离纯化,对不同时段的收集液做抑菌试验,以大肠杆菌O78标准菌株为指示菌,采用打孔法进行抑菌活性检测,比较评价分步层析效果,以Tricine-SDS-PAGE进行分子质量检测。结果显示,通过5和3 ku卷式膜超滤获得的3~5 ku组分蛋白质样品抗菌活性较强;对于3~5 ku组分经凝胶过滤层析分离纯化,纯化后的抗菌肽A3抑菌活性最强,经Tricine-SDS-PAGE小分子多肽电泳检测,已达到电泳纯,分子质量为4 ku;抑菌活性检测结果显示,该抗菌肽A3对大肠杆菌O78标准菌株具有较强的抑菌作用。同时,抗菌肽A3表现出较好的耐热性,90~100 ℃处理15 min,抑菌活性可保持在96%左右;具有较好的酸碱稳定性,在pH 2.0~9.0下,抑菌活性保持在90%以上;经胃蛋白酶作用后抗菌肽A3抑菌活性降低20%,胰蛋白酶作用后抗菌肽A3抑菌活性降低18%,蛋白酶K对抗菌肽A3的抑菌活性几乎无影响。本研究结果表明,分离得到的抗菌肽A3是一种对大肠杆菌O78具有抑菌活性的新型抗菌肽,具有一定的开发潜力,可为下一步抗菌肽的结构分析、理化特性分析等深入研究提供一定参考依据。     

Identification of an antihypertensive peptide derived from chicken bone extract

A novel angiotensin‐converting enzyme (ACE) inhibitory peptide was isolated and purified from chicken bone extract by enzymatic digestion. The peptide was defined as an ACE inhibitor, and it demonstrated antihypertensive activity following oral administration to spontaneously hypertensive rats (SHRs). The results of this study suggest that peptides derived from an extract of chicken bones, administered orally, have the ability to reduce the blood pressure of SHRs significantly over a short period of time (3 h). Moreover, the blood pressure then remains low for 3 h. This peptide derived from chicken bones may therefore have great value as a short‐term remedy for chronic conditions such as high blood pressure. The amino acid sequence of the peptide was YYRA (Tyr‐Tyr‐Arg‐Ala), which was the origin of the Ig heavy chain V region (27–30 position). The IC₅₀ value of its synthetic peptide was 33.9 μg/mL. We suggest that the ACE inhibitory and antihypertensive peptides derived from chicken bone extract may contribute to develop physiologically functional foods or improve food functionality.     

Partial purification and characterization of chicken interleukin-2.

Chicken interleukin 2 (IL-2) activity was partially purified from conditioned medium produced by culturing chicken splenic lymphocytes in the presence of concanavalin A. The purification procedure included sequential steps of gel filtration chromatography, reverse-phase high-pressure liquid chromatography, and phenyl-sepharose chromatography. Two peaks of IL-2 activity with apparent mol. wt. ranges of 36-39 kD and 17.5-25 kD were eluted from the Sephadex G100 gel filtration column. An increase in IL-2 spec. act. from 14 U mg-1 to between 2000 and 20,000 U mg-1 was obtained for the Sephadex G100 column peaks when subjected to the subsequent steps of the purification procedure. Alkylative reduction of the higher mol. wt. Sephadex G100 column peak (followed by re-chromatography with Sephadex G100), resulted in generation of the lower (17.5 kD) mol. wt. peak, indicating that chicken IL-2 is capable of either dimerizing or forming aggregates with other proteins. Elution of the lower mol. wt. IL-2 activity from a non-reducing sodium dodecyl sulfate-polyacrylamide gel demonstrated an apparent mol. wt. for chicken IL-2 of 20 kD, which confirmed the range of 17.5-25 kD seen with gel filtration.     

Studies on Aspergillus Fumigatus; Hydrolysis of Polyamino Acids by Mycelial Filtrate and Chromatographic Fractionation of the Filtrate

Mycelial filtrates from Aspergillus fumigatus (AF), shown to possess haemolytic, toxic, casein precipitating, and protein hydrolyzing activity, hydrolyzed poly-L-lysine and poly-L-glutamine in the pH range 4.6—5.3. Incipient activity against poly-L-lysin was observed at pH 9. Owing to spontaneous hydrolysis of the polyamino acid at pH > 10, no activity optimum could be traced.Gel filtration of mycelial filtrate on Sephadex G-75 or G-100 columns offered no definite information whether the protein hydrolyzing activity, using haemoglobin as substrate, at the optimum pH values, 2.9, 4.6 and 10, shows the activity of a single enzyme with more than 1 pH optimum or of more than 1 enzyme active at different pH values. Certain results of the investigations seem to indicate that the protein hydrolyzing activity at pH 2.9 was not caused by enzymes identical with the enzyme (s) causing the protein hydrolyzing activity at pH 4.6 or pH 10.Casein precipitating and protein hydrolyzing activity occurred, following gel filtration on a Sephadex G-100 column, in identical fractions whereas neither haemolysin nor toxin could be found in samples of 0.5 ml fraction solution from any of the fractions after filtration on Sephadex G-75 or G-100 columns.By using antiserum to a crude filtrate from a homologous AF strain it could be shown, applying immuno-electrophoresis, that dialyzed mycelial filtrate contained 8 precipitating antigens whereas proteinase purified by gel filtration and displaying protein hydrolyzing activity at pH 2.9, pH 4.6 and pH 10 contained 4 such antigens.     

兔腰大肌和鸡胸大肌三聚磷酸酶特性的比较研究

分离纯化兔腰大肌和鸡胸大肌中的三聚磷酸盐酶(TPPase),比较不同物种TTPase的酶学特性和作用条件。试验结果,显示兔腰大肌和鸡胸大肌的TPPase的最适温度分别为35℃和30℃;最适pH分别是6和5。Mg2+对兔腰大肌和鸡胸大肌的TPPase都有激活作用,在0~20 mmol/L范围内,鸡胸大肌TPPase活性随Mg2+浓度增加而缓慢上升,3 mmol/L的Mg2+对兔腰大肌TPPase激活作用最明显。Ca2+对兔腰大肌TPPase有激活作用,而对鸡胸大肌TPPase有抑制作用。高浓度的EDTA-Na4和KIO3对两种肉TPPase都有明显的抑制效果。EDTA-Na2对兔腰大肌TPPase的活性没有显著影响,但在高于0.5 mmol/L时,对鸡胸大肌TPPase的活性抑制效果明显。     

麻疯树叶提取物对鸡大肠杆菌、金黄色葡萄球菌的体外抑菌作用

本试验研究了麻疯树叶的乙醇提取物对鸡常见致病性菌:大肠杆菌、金黄色葡萄球菌的体外抑菌活性。结果显示:该提取物对鸡大肠杆菌、金黄色葡萄球茵均有抑制作用,对大肠杆菌的抑菌作用优于对金黄色葡萄球菌。细菌接种量对麻疯树叶提取物抑制鸡大肠杆菌的作用无明显影响,但接种量升至107cfu／mL,使其对金黄色葡萄球菌的MIC增加1倍。碱性条件可增强其抗菌活性。     

Characteristics of a haemolytic extract from avian Pasteurella multocida

In experimental fowl cholera, the intramuscular inoculation of Pasteurella multocida induces tissue damage that implies proteolytic or cytolytic activity of the bacteria. Such activity could not be demonstrated by conventional in vitro tests. The treatment of P. multocida strain VP21 with Tween-80 yielded an extract that lysed washed chicken red cells. Extracts were active to a maximum titre of 64. Haemolytic activity of the extract was neither affected by boiling nor by extremes of pH, indicating the active component was not a simple protein. Treatment with trypsin had no effect, but it was inactivated by Proteinase K. Yields were highest from bacteria grown in dextrose starch- or casein sucrose-yeast broths; were similar if cultured in air or anaerobically, but were reduced if the bacteria were grown in 5% CO(2). Haemolytic activity was eliminated on exposure to serum or serum albumen. The extract from strain VP21 haemolysed red cells from the chicken, rabbit, sheep, horse, bovine and human, with the highest titres observed on chicken cells. Six other avian strains and seven out of 10 strains of P. multocida from other species yielded an extract which haemolysed chicken red cells. The elaboration of this cytotoxic substance in vivo and its role in pathogenesis remains to be determined.     

合成绵羊抗菌肽NK-Lysin抗菌活性及其对雏鸡沙门氏菌攻毒的治疗效果研究

为了研究绵羊抗菌肽NK-Lysin主要活性区的生物学功能,试验设计并合成了3对功能区多肽片段,并利用径向扩散试验和最小抑菌浓度对其抗菌活性进行检测,分析了合成多肽对鸡血红细胞的毒性作用,筛选出抗菌效果最好的多肽对沙门氏菌攻毒的雏鸡进行治疗,检测其治疗效果。结果表明,合成多肽对大肠杆菌、沙门氏菌具有抑制活性,多肽片段长度、多肽C-端有无酰胺化及多肽内的二硫键是否成环对多肽的抗菌活性均有影响;筛选出的2个多肽在治疗雏鸡沙门氏菌攻毒的过程中能够明显降低死亡率,对雏鸡心脏、肝脏、肾脏的病理损伤也明显小于对照组。本研究结果为绵羊抗菌肽NK-Lysin作为候选抗菌药物的开发奠定了基础。     

Antimicrobial Activity of Synthesized Sheep NK-Lysin Peptides and Its Treatment for Chicken Salmonella Challenge

In order to study the biological function of the main active region of sheep antimicrobial peptide NK-Lysin, three pairs of functional domain peptide fragments were designed and synthesized the antibacterial activity was detected by radial diffusion test and minimal inhibitory concentration. We analyzed the toxic effects of chicken red blood cells and screen the best peptides for treatment chicken challenged by Salmonella pullorum in this study. The rusults showed that synthesized peptides were inhibitory to Escherichia coli and Salmonella pullorum. Fragment length of peptides, C-terminal amidation and peptide inner loop were essential to antibacterial activity. Two of synthesized peptides were used to treat chichen challenged by Salmonella pullorum which obviously decreased the mortality of chicken. The pathological damage of heart,liver,kidney were less than that of control group. This study laid a foundation for the development of the sheep NK-Lysin peptides as candidate antimicrobial agents.