光滑爪蟾皮肤抗菌肽的分离纯化及活性检测

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。     

蜜蜂幼虫抗菌活性的初步研究

为了研究中华蜜蜂幼虫粗提取液的抗菌活性,试验通过诱导2日龄蜜蜂幼虫,分离、纯化出中华蜜蜂幼虫中的抗菌肽,研究其性质和抑菌特性。结果表明:中华蜜蜂幼虫提取液中含有抑菌活性物质,对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、藤黄八叠球菌均有明显的抑制作用,而对酵母和霉菌的生长没有明显抑制作用;此外,中华蜜蜂幼虫粗提取液分别经过高温、高盐、酸碱处理后仍保持较高的抑菌活性。说明蜜蜂幼虫的抑菌活性物质含有抗菌肽。     

绵羊抗菌肽GNLY基因多态性分析及合成多肽的生物学功能研究

试验旨在分析绵羊抗菌肽颗粒溶素（granulysin,GNLY）基因多态性,检测合成多肽的抑菌活性和抑制肿瘤细胞活性,明确绵羊抗菌肽GNLY的生物学功能。通过对GNLY基因RT-PCR产物进行测序,分析GNLY基因多态性,推导其氨基酸序列信息合成功能区多肽,利用径向扩散试验和MIC试验检测合成多肽对大肠杆菌、沙门氏菌、葡萄球菌、金黄色葡萄球菌的抑制作用;利用MTT试验观察合成多肽对人食管癌细胞（EC109）、人肾癌细胞（X786-0）的抑制作用。结果发现,MIC试验中浓度>250 μg/mL的G16对大肠杆菌、葡萄球菌均有抑制作用;浓度 ≥ 7.82 μg/mL的GS16对大肠杆菌抑制作用明显,对沙门氏菌、葡萄球菌和金黄色葡萄球菌无明显抑制作用;62.5 μg/mL GC16对大肠杆菌、沙门氏菌和金黄色葡萄球菌均有抑制作用,其中对大肠杆菌和金黄色葡萄球菌抑制作用较为明显;G22对4种细菌均无明显的抑制作用。MTT试验结果表明,合成多肽G16、GS16对癌细胞无抑制作用,G22和GC16对EC109、X786-0具有显著抑制作用,其中1 mg/mL G22和1 mg/mL GC16对EC109生长抑制率分别为88.21%和57.21%,对X786-0生长抑制率分别为96.37%和81.87%。研究显示,合成GNLY多肽对细菌和肿瘤细胞具有一定的抑制活性,本研究结果为绵羊抗菌肽作为候选抗菌药物的开发奠定了基础。     

绵羊抗菌肽GNLY基因多态性分析及合成多肽的生物学功能研究

试验旨在分析绵羊抗菌肽颗粒溶素(granulysin,GNLY)基因多态性,检测合成多肽的抑菌活性和抑制肿瘤细胞活性,明确绵羊抗菌肽GNLY的生物学功能。通过对GNLY基因RT-PCR产物进行测序,分析GNLY基因多态性,推导其氨基酸序列信息合成功能区多肽,利用径向扩散试验和MIC试验检测合成多肽对大肠杆菌、沙门氏菌、葡萄球菌、金黄色葡萄球菌的抑制作用;利用MTT试验观察合成多肽对人食管癌细胞(EC109)、人肾癌细胞(X786-0)的抑制作用。结果发现,MIC试验中浓度250μg/mL的G16对大肠杆菌、葡萄球菌均有抑制作用;浓度≥7.82μg/mL的GS16对大肠杆菌抑制作用明显,对沙门氏菌、葡萄球菌和金黄色葡萄球菌无明显抑制作用;62.5μg/mL GC16对大肠杆菌、沙门氏菌和金黄色葡萄球菌均有抑制作用,其中对大肠杆菌和金黄色葡萄球菌抑制作用较为明显;G22对4种细菌均无明显的抑制作用。MTT试验结果表明,合成多肽G16、GS16对癌细胞无抑制作用,G22和GC16对EC109、X786-0具有显著抑制作用,其中1mg/mL G22和1mg/mL GC16对EC109生长抑制率分别为88.21%和57.21%,对X786-0生长抑制率分别为96.37%和81.87%。研究显示,合成GNLY多肽对细菌和肿瘤细胞具有一定的抑制活性,本研究结果为绵羊抗菌肽作为候选抗菌药物的开发奠定了基础。     

苦马豆素对人食道癌Eca-109细胞体外生长的抑制试验

探讨苦马豆素 (SW)对人食道癌 Eca-10 9细胞生长的抑制作用 ,以揭示 SW治疗食道癌的作用机制。选用不同剂量的 SW与食道癌Eca- 10 9细胞共同培养不同时间后 ,用 MTT法观察 SW对食道癌 Eca- 10 9细胞生长的抑制作用 ;细胞 DNA经特异性荧光染色后 ,用流式细胞仪分析细胞周期变化。结果 :SW对食道癌Eca- 10 9细胞生长的半数抑制浓度 IC50 <2 .5μg/ m L;细胞周期分析 ,Eca- 10 9细胞 G1期细胞增多 ,S期细胞减少。结果表明 :SW可通过抑制人食道癌 Eca- 10 9细胞系生长达到抑制肿瘤的目的。     

抗菌肽BSN-37对大肠杆菌胞内物质泄露的影响

为了解抗菌肽BSN-37对大肠杆菌的作用机制,通过β-半乳糖苷酶试验测定了抗菌肽BSN-37对大肠杆菌CVCC1568的抑菌活性,以菌落计数法测定了抗菌肽BSN-37对大肠杆菌CVCC1568的抑菌动力学,用分光光度法测定了抗菌肽BSN-37对大肠杆菌CVCC1568细胞壁通透性,以及胞内紫外吸收物质、蛋白及核酸的泄露量。结果表明,抗菌肽BSN-37能有效抑制大肠杆菌CVCC1568的生长繁殖,随抗菌肽浓度增加,抑制活性越高,抗菌肽BSN-37作用后的大肠杆菌CVCC1568细胞壁通透性增加,胞内紫外吸收物质、蛋白和核酸的泄露量随时间延长而逐渐增加。说明抗菌肽BSN-37能破坏大肠杆菌CVCC1568的细胞壁和细胞膜,从而表现较好的抑菌活性。     

抑霉乳酸菌的筛分及其抑菌特性研究

试验旨在获取生物抑霉的优良菌株以及为质量安全青贮饲料生产提供高效菌剂。从青贮饲料、酸奶以及奶豆腐中分离纯化出乳酸菌和霉菌,并且通过双层平板法对具有抑制霉菌活性的乳酸菌菌株进行筛选,测定其上清液抑霉效果;采用蛋白酶处理和高温处理法分析乳酸菌抑霉的有效成分。结果表明:分离筛选的乳酸菌和霉菌经鉴定为植物乳杆菌和黄曲霉菌,且植物乳杆菌对黄曲霉菌的生长有明显抑制作用。蛋白酶处理对乳酸菌无细胞上清液的抑菌活性有不同影响,而加热处理并不改变其抑菌效果。研究表明,植物乳杆菌对黄曲霉菌生长有明显的抑制作用,推测其抑菌活性物质为蛋白质、肽类等,并且这些抑菌活性物质的热稳定性较高。     

人工设计天蚕素抗菌肽AMP-D及其活性测定

本试验通过对抗菌肽结构改造优化,得到了一条新设计的抗菌肽AMP-D,并利用固相合成技术人工合成后对其活性和溶血性进行测定。试验结果表明,AMP-D对革兰氏阴性菌及真菌没有生物活性,对革兰氏阳性菌有明显的抑制作用,且其没有溶血活性;AMP-D对金黄色葡萄球菌的最小抑菌浓度为10.5 μg/mL,较原设计肽活性有较大提高。     

蛙源抗菌肽Magainin Ⅱ对畜禽常见病原菌抑菌活性与体外稳定性测定

抗菌肽MagaininⅡ是来源于非洲爪蟾体内的一种具有抗菌活性的小肽类物质。试验采用微量肉汤稀释法测定蛙源抗菌肽MagaininⅡ对几种常见畜禽致病菌的抑菌活性;在此基础上将抗菌肽MagaininⅡ在不同温度、pH值及人工胃蛋白酶与胰蛋白酶进行处理,进而评价其稳定性。试验结果表明,抗菌肽MagaininⅡ对革兰氏阳性菌不具有抑菌作用,但对几种革兰氏阴性菌表现出一定的抗菌活性,MIC值处于64~256μg/ml;由时间杀菌曲线可知,MagaininⅡ不能有效抑制金黄色葡萄球菌ATCC 25923菌落的形成,但能够在90 min内对大肠杆菌ATCC 25922发挥抑制作用;MagaininⅡ的稳定性测试结果表明,分别经不同温度、pH值以及人工胃蛋白酶与胰蛋白酶处理后的MagaininⅡ对金黄色葡萄球菌ATCC 29523和表皮葡萄球菌ATCC 12228始终没有发挥抑菌活性,在经50~70℃处理后能够对大肠杆菌K12和肠炎沙门氏菌CMCC 50041稳定发挥抗菌效果,MIC值始终为64μg/ml;经两种人工酶处理后的MagaininⅡ对两种试验革兰氏阴性菌不再具有抑菌作用;经pH值为2处理后,MagaininⅡ对大肠杆菌K12和肠炎沙门氏菌CMCC 50041的MIC均为256μg/ml,比未处理时明显增大,经pH值为8处理后,MagaininⅡ不能抑制大肠杆菌K12,对肠炎沙门氏菌CMCC50041的MIC为128μg/ml。基于试验结果可推断:MagaininⅡ对革兰氏阳性菌抗菌效果不明显,对革兰氏阴性菌具有一定的抗菌效果,经人工酶处理后性质不稳定,该研究为MagaininⅡ在畜禽生产上的应用提供参考。     

新疆家蚕抗菌肽（Cecropin-XJ）对奶牛乳房炎病原的体外抑菌效果观察

利用新疆家蚕抗菌肽对奶牛乳房炎病原菌的体外抑菌试验进行药效学观察,确定抗菌肽的抗菌谱,为今后选择新药提供资料和依据。挑选乳房炎的主要病原菌进行药敏试验。先确定抗菌肽的最小抑菌浓度。再将抗菌肽和抗生素进行比较。家蚕抗菌肽对奶牛乳房炎常见病原菌中的革兰氏阳性球菌生长有抑制作用,而对大肠杆菌没有生长抑制作用;与抗生素抑菌圈相比,家蚕抗菌肽抑菌圈较小。结果提示抗菌肽没有抗生素抑菌效果强,但抗菌肽可以减少耐药性,为今后抗菌肽与抗生素联合用药提供依据。     

乳清蛋白抗菌肽的分离纯化及活性

以乳清蛋白为原料,酶解制备具有抗菌能力的生物活性肽。采用超滤、葡聚糖凝胶层析色谱、反相高效液相色谱法对酶解液进行分离纯化,并对分离产物的氨基酸组成进行分析。结果表明:葡聚糖凝胶色谱纯化最佳流速2 mL/min,最佳样品质量浓度15 mg/mL;再由反相高效液相色谱分离的高活性抗菌肽,抑菌圈直径达到31.33 mm。氨基酸分析结果显示,高活性抗菌肽碱性氨基酸和疏水性氨基酸含量最多,分别为42.69%和37.39%。     

Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase

This study aims to identify peptides with angiotensin-I converting enzyme (ACE) inhibitory activity in hydrolysate from chicken leg bone protein hydrolyzed with alcalase for 4 h (A4H). The hydrolysate has demonstrated potent in vitro ACE inhibitory activity, and has been shown to attenuate the development of hypertension and cardiovascular hypertrophy in spontaneously hypertensive rats (SHR). A4H is competitive for ACE and was separated using high-performance liquid chromatography (HPLC) with a gel filtration column (Superdex Peptide HR 10/30). The results show that A4H is a mixed non-competitive inhibitor. Eighteen fractions were detected after separation of A4H, and most of them showed ACE inhibitory activity. Five fractions with strong ACE inhibitory activities (above 50%) were labeled from A to E. In addition, there were 10 peptides, consisting of 5–10 amino acid residues that were identified from fraction D that exhibited the strongest ACE inhibitory activity. Three of the identified peptides corresponded to peptides derived from collagen type I and chicken muscular protein. It is revealed that A4H has several peptides that possess ACE inhibitory activities.     

Gel filtration of lignosulphonic acids and peptide-precipitating abilities of the separated fractions

Lignosulphonic acids in dialysed sulphite spent liquor and purified lignosulphonic acids were subjected to gel chromatography on Sephadex G-75, G-100 and G-200 and the fractions tested for peptide-precipitating ability. About 56 % of the total lignosulphonic acids in the dialysed sulphite spent liquor had estimated molecular weights above 90000 and about 72 % above 44000. About 94 % of the purified lignosulphonic acids had molecular weights above 90000 and the remaining 6 % had above 36000. The major peptide-precipitating activity of the lignosulphonic acids was due to fractions with molecular weights in excess of 90000. The percentage of peptides in the peptide-lignosulphonic acid precipitates was found to be 80–90. The molecular weights of the peptides used were found to have an upper limit of about 20000. The lower limit for molecular weights of lignosulphonic acid-precipitating peptides is estimated to be below 6000.Keyword: gel filtration, lignosulphonic acids, molecular weight, peptide-precipitating ability     

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。     

Isolation and characterization of C-reactive protein and serum amyloid P component from bovine serum

C-reactive protein (CRP) and serum amyloid P component (SAP), which are known as acute phase reactants in human and many other animals, were purified from cow sera. Affinity chromatography using HE agarose gel was the most effective method to isolate both CRP and SAP from a large volume of bovine serum. Separation of CRP and SAP from the mixed preparation could be performed by DEAE-cellulose column chromatography, gel permeation HPLC using TSK-G3000SW or affinity chromatography using phosphorylcholine derivatives of bovine serum albumin-conjugated Toyopearl HW 65. Bovine CRP and SAP were identified as genuine CRP- and SAP-class proteins by their cross reactivities with anti-human CRP and anti-human SAP, respectively, and by their homology in amino acid compositions compared with those of human CRP and SAP, respectively. Bovine CRP moved slower than beta-globulin, and bovine SAP moved in the beta-globulin region in agarose gel electrophoresis. Both of them gave single bands in native polyacrylamide gel electrophoresis (PAGE). Bovine CRP and SAP molecular weights were estimated to be 100,600 and 109,500 daltons respectively, by sedimentation equilibrium analysis. Bovine CRP showed 23K dalton subunits by sodium laurylsulfate-PAGE and bovine SAP showed 28K and 32K dalton subunits, both of which were glycosylated and had identical amino acid compositions, indicating that both CRP and SAP molecules are pentamers. In fact, they appeared to have pentameric disk-like configurations in electronmicroscopical examination.     

Partial purification and characterization of a protein in porcine follicular fluid which restricts sperm-egg interaction in vitro.

An attempt was made to isolate and characterize a component in preovulatory porcine follicular fluid (pFF) which has a restricting effect on sperm-egg interaction in vitro. Using the zona-free hamster ova (eggs) penetration assay as an in vitro test system, it was shown previously that the numbers of porcine spermatozoa attached to or penetrated into each egg and the number of eggs with sperm attached or penetrated decreased significantly as the concentration of pFF was increased in the culture medium. In the present study, the component in pFF having these effects was shown to be a heat stable, nonsteroidal substance which retained its activity after dialysis, lyophilization and gel filtration chromatography. The activity was also found to be present in preovulatory homologous serum. Separation of the material on protein type gel filtration columns with detection at 280 nm, together with the banding seen with Coomassie staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggests that it is a protein. Based on high pressure liquid chromatographic separation (HPLC) and SDS-PAGE analyses, the bioactivity could be due to a single protein of 87 kD or to one or more of three smaller proteins, possibly disaggregated products of the 87 kD protein, in the range of 26-28 kD.     

Staphylococcosis of turkeys. 4. Characterization of a bacteriocin produced by an interfering Staphylococcus

A Staphylococcus epidermidis isolate designated strain 115, which is used as an interfering agent against staphylococcosis of turkeys, produces a bacteriocin that was partially purified and characterized in this study. This bacteriocin diffused through agar media, but it was not found in appreciable quantities in the supernatant fluid of broth cultures. Extraction of the bacterial cells with 7 M urea, 1% sodium dodecyl sulfate, or 1% Triton X-100 caused considerable amounts of the bacteriocin to go into solution. This substance was partially purified by selective chemical extraction and by gel filtration chromatography using a Sephacryl S-300 column. This bacteriocin had two active forms: an aggregate, and a small-molecular-weight form estimated by gel filtration chromatography to be less than 6500. Activity was not affected by heat, repeated freeze-thaw cycles, pH 2 and pH 10, or a variety of proteolytic enzymes, nucleases, a lipase, and lysozyme.     

Unsaturated vitamin b(12) binding capacity in human and ruminant blood serum - a comparison of techniques including a new technique by high performance liquid chromatography

A new technique by High Performance Liquid Chromatography (HPLC-gel permeation) shows promise as a tool to separate and quantitate the Unsaturated Vitamin B(12) Binding Capacity (UBSC) of the individual Vitamin B(12) binders in blood serum. This method, although not as rapid as protein-coated charcoal or cellulose separation techniques, is more applicable for use with large numbers of samples than gel filtration. The use of a radioactivity detector to monitor the eluant from the column permitted automation of the method. Comparable results for UBBC and for the UBBC of individual binders were obtained when samples were analyzed by gel filtration and HPLC. The HPLC method proved suitably precise and the recovery of added cyanocobalamin was acceptable. It is proposed that HPLC be the method of choice for measurement of the USBC of binders of Vitamin B(12) in blood serum.     

The antigenic activity of chromatographic fractions of a saline extract of Taenia saginata (Goeze, 1782) proglottides

A saline extract of a homogenate of Taenia saginata proglottides was partially purified by gel filtration chromatography on Sephadex G200 or Sepharose 4B and by ion exchange chromatography on DEAE cellulose. Gel filtration produced two distinct fractions with different antigenic properties. The first was of molecular weight of approximately 1,000,000 and contained a high level of activity in the haemagglutination inhibition test. The second fraction of molecular weigh of approximately 100,000 contained most of the immuno-precipitin activity. Other fractions had little or no antigenic activity. Eight fractions were obtained by DEAE cellulose chromatography, of which 4 had detectable antigenic activity. Subsequent rechromatography of fractions obtained by gel filtration on DEAE cellulose produced relatively pure fractions of high antigenic activity, from which small molecular weight contaminants had been removed.