高效液相色谱分离胸腺免疫抑制提取物高活性组分1及分离组分的性质

用高效液用色谱对猪胸腺免疫抑制提取物高活性组分1进行了分离，得到三个组分，所得组分H1、H2、H3在10μg／ml浓度下对植物凝集素诱导的人外周血淋巴细胞转化抑制率分别为59．95％、47．61％和73．93％。各组分的紫外光谱表明，活性并不象以往报道的那样与核酸含量有关，而显示活性与A260／A280值无规律性关系。     

高效液相色谱分离胸腺免疫抑制提取物高活必组分1及分离组？…

用高效液相色谱对猪胸免疫抑制提取物高活性组分１进行了分离，得到三个组分，所得组分Ｈ１、Ｈ２、Ｈ３在１０μｇ／ｍｌ２下对植物凝集素诱导的人外周血淋巴细胞转化抑制剂分别为５９．９５％、４７．６１％和７３．９３％。各 分的紫外光谱表明，活性并不象以往报道的那样与核酸含量有关，而显示活性与Ａ２６０／Ａ２８０值无规律性关系。     

诃子抗CPG ODN组分的分离及活性评价

目的：从诃子中分离制备具有拮抗CPGODN作用的活性组分。方法：应用水提、离子色谱技术等方法，从诃子中分离制备具有拮抗CPGODN活性的组分；并应用生物传感器技术，对所得到的组分进行与CPGODN结合活性的筛选；观察其对CPGODN刺激的小鼠RAW264.7细胞释放炎症介质的抑制作用。结果：应用生物传感器技术筛选出与CPGODN结合活性最高的诃子作为研究对象，应用离子色谱技术从诃子中分离出3组分，其中有3个组分均具有一定的与Lipid A的结合活性，以第3组分与Lipid A的结合活性最强；并对CPGODN刺激的小鼠RAW264．7细胞释放TNF-α具有显著的抑制作用；结论：从诃子中分离出的第3组分具有显著的拮抗CPGODN活性。     

马鹿茸血疏水性肽的分离及组分3的免疫活性

采用大孔吸附树脂和凝胶色谱等方法,从马鹿茸血蛋白酶解产物中分离纯化获得免疫活性肽.通过体外试验研究其免疫活性.结果表明:大孔吸附树脂可依据氨基酸的疏水性对肽段进行分离,经梯度洗脱后,其中用75%乙醇洗脱所得的组分促脾淋巴细胞增殖活性最高.纯化所得的产物中组分3具有较高的DPPH自由基清除能力,可刺激白介素-1的分泌、促进巨噬细胞的吞噬活性.     

蜈蚣胃蛋白酶解物体外抗凝活性肽类的分离与分析

目的采用凝胶过滤色谱和C18反相色谱对蜈蚣胃蛋白酶解物的抗凝活性部分进行分离和纯化,并检测其相对分子质量分布范围。方法光谱法收集各凝胶色谱并分离各流分;用活化部位的凝血活酶时间(APTT)作为指标,检测各流分体外的抗凝活性;用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)检测其相对分子质量的分布范围。结果经过滤色谱和C18反相色谱纯化后得到了抗凝活性成分,并测得成分中的相对分子质量的分布范围在568~1126之间。结论凝胶过滤色谱和C18反相色谱对蜈蚣胃蛋白酶解物的抗凝活性部分进行分离和纯化后,抗凝活性组分是小分子肽类。     

高压液相色谱技术分离制备杆菌肽各组分的研究

目的 开发制备型高压液相色谱分离杆菌肽各组分的方法.方法 根据杆菌肽各组分化学结构性质,采用纳微UnisilC1s柱,通过改变流动相pH和流动相配比,以及不同的上样量,探索杆菌肽各组分分离的方法.收集分离得到的不同组分,经脱盐-冷冻干燥,得到相应杆菌肽组分冻干粉.结果 纳微Unisil C18柱可以用于杆菌肽各组分的分离,流动相配比为甲醇(A)∶[1.54‰乙酸铵水溶液(乙酸调pH至5.0)-乙腈(9∶1,V/V)] (B)=9.0∶ 11.0(V/V),总流速40 mL· min-1,上样量200mg,检测波长为254 nm.制备得到的杆菌肽各组分纯度均大于95％.结论 该方法上样量大,稳定性好,收集到的各组分纯度高,为杆菌肽各组分的分离制备提供了借鉴.     

镇痛活性肽VRD突变体(S54A)的构建与表达以及对活性的影响

目的研究54位丝氨酸对镇痛活性肽(analgesic peptide,AGP)VRD生物活性的影响。方法利用一步聚合酶链式反应(polymerase chain reaction,PCR),用Ala替代镇痛活性肽VRD第54位的Ser,从而构建突变体S54A。采用本实验室构建的pSYPU作为表达载体在大肠杆菌BL21(DE3)中表达重组蛋白。通过金属离子螯合亲和柱色谱和阳离子交换柱色谱方法对重组蛋白进行分离纯化。小鼠扭体法测定重组蛋白的镇痛活性。结果重组蛋白主要以可溶性形式存在,通过两步色谱方法获得了电泳纯样品。突变体S54A的镇痛活性明显低于AGP-VRD(P<0.05)。结论镇痛活性肽VRD中54位丝氨酸与其镇痛活性密切相关。     

猪胸腺免疫抑制组分的生化性质和免疫活性

以猪胸腺为原料获得免疫抑制提取物,经超滤分离得到具较高活性的组分 U_2。U_2对机体细胞免疫和体液免疫均有显著的抑制作用,并能显著延长大白鼠移植皮肤的存活期。U_2经凝胶色谱得到分子量约10000的富含酸性氨基酸的具更高活性的组分 P_3,其在高效液相色谱中可见有两个洗脱峰。所有组分均含有多肽和核酸。     

海洋青铜小单孢菌FIM 02-523产生的脂肽类化合物FW523的分离鉴别和生物学活性

在筛选新免疫抑制剂的过程中，从海洋青铜小单孢菌Micromonospora chalcea FIM 02-523发酵液提取到脂肽类化合物FW523，经纯化得到5个组分。组分3（FW523-3）与抗肿瘤抗生素rakicidin B同质，但它具有与紫杉醇相当的抗肿瘤活性和与环孢菌素相当的免疫抑制活性。组分1和2（FW523-1，2）与rakicidin A分子量相同，组分4（FW523-4）比rakicidin B多1个-CH2，组分5（FW523-5）比rakicidin A少1个-CH2，它们是rakicidins的两个新同系物。     

赤芍拮抗内毒素活性物质的分离及生物学活性研究

目的 通过生物传感器技术，应用常规分离技术寻找赤芍中的非鞣质类的抗LPS活性成分。方法 以生物传感器技术和薄层色谱技术为指导，建立赤芍抗LPS成分的有效提取方法。再利用高效液相色谱技术（HPLC）从赤芍中定向分离出具有拮抗LPS作用的活性组分。最后对其进行拮抗LPS活性的药理学评价。结果 利用生物传感器技术完成了对不同产地和不同煎煮方法的赤芍拮抗LPS活性的筛选，确定出醇提法的四川的赤芍中拈抗LPS活性成分的含量最高。并从中定向分离出6个具有与Lipid A结合活性的组分。     

Isolation and some biochemical properties of a paralysing toxin from the venom of the wasp Microbracon hebetor (Say)

A toxin with paralysing activity was isolated from crude preparations of Microbracon hebetor (Say) venom by ion exchange chromatography on DEAE-Sephadex A-50, gel chromatography on Sephadex G-100, electrophoresis on polyacrylamide gel and gel chromatography on Sephadex G-75. The active component is very labile. A 28-fold purification was obtained with a recovery of about 2.5% of the original biological activity. The toxin shows one single protein band after disc electrophoresis. The molecular weight was assessed at 61,000 by gel chromatography, and at 62,700 by the sedimentation equilibrium method. The isoelectric point was at pH 6.8. The purified toxin was inactivated by a number of proteolytic enzymes.     

浙江蝮蛇(Agkistrodon halys Pallas)蛇毒蛋白水解酶的分离纯化及其理化性质研究

应用DEAE-Sephadex A-50,Sephadex G-75,Sephadex G-200和QAE-SephadexA-25柱层析,从浙江蝮蛇毒中分离纯化出一种具有酪蛋白水解活性的蛋白水解酶a,称之为蛋白酶a.纯化后的蛋白酶经聚丙烯酰胺凝胶电泳和等电聚焦电泳鉴定,均呈现一条区带。等电点为6.4.用凝胶过滤及SDS-聚丙烯酰胺凝胶电泳测得其分子量为68000,糖蛋白染色法表明蛋白酶a是一糖蛋白。紫外吸收光谱表明蛋白酶a在277nm波长处有最大吸收,消光系数E_(277nm)~(0.1%)=0.681.蛋白酶a含有丰富的天冬氨酸、谷氨酸和赖氨酸。     

Studies on auromomycin.

A new antitumor antibiotic, named auromomycin, was isolated from the culture broth of Streptomyces macromomyceticus, a macromomycin-producing strain. The antibiotic was recovered from the culture filtrate by salting out with ammonium sulfate and further purified by successive application of ion-exchange chromatography on Amberlite IRA-93 (Cl form) and DEAE-Sephadex (OH form), Gel filtration on Sephadex G-50 and hydrophobic chromatography on Octyl-Sepharose CL-4B. The antibiotic is an acidic polypeptide with a molecular weitht of 12,500 and an isoelectric point of pH 5.4 and consists of 16 different amino acids. It has characteristic absorption maxima at 273 nm and 357 nm in the ultraviolet spectrum and two minima at 280 nm and 350 nm in the optical rotatory dispersion spectrum. Auromomycin exhibits antibacterial activity not only against Gram-positive bacteria, but also Gram-negative bacteria. Antitumor activities of auromomycin were revealed against EHRLICH ascites carcinoma, ascites sarcoma 180, L1210 leukemia and LEWIS lung carcinoma. Auromomycin was found to be converted into macromomycin by adsorption chromatography on Amberlite XAD.     

羊小肠抗菌肽分离及其生物活性研究

目的:从羊小肠提取抗菌肽并对其抑菌作用进行研究,寻找廉价易得的广谱高效天然抗菌肽。方法:葡聚糖凝胶分离及反相高效液相色谱法提纯羊小肠活性抗菌肽;采用琼脂平板打孔法进行抑菌效果的测定;SDS-PAGE电泳分析相对分子质量。结果:反相高效液相色谱法分离出现3个主峰,其中之一对大肠杆菌和金葡球菌有较高抑菌活性(蛋白浓度分别为38,19,10 m.gmL-1);相对分子质量约为40 Ku。结论:实验得到的抗菌肽材料易得,对G-菌和G+菌均有较强的抑制作用。     

Characterization of the anticoagulants from Taiwan cobra (Naja naja atra) snake venom

Taiwan cobra (Naja naja atra) snake venom was separated into 19 fractions by means of CM-Sephadex C-50 column chromatography. Anticoagulant Fractions V-VII were refractionated by gel filtration on Sephadex G-50 and the purified component possessed phospholipase A2 activity and an inhibitory effect on collagen-induced platelet aggregation. The anticoagulant action could be antagonized by phospholipid or platelet factor 3. Anticoagulant Fraction XVII was also further refractionated by gel filtration on Sephadex G-50 and the purified component was shown to be cardiotoxin. It was a weak anticoagulant, caused direct hemolysis and potentiated collagen-induced platelet aggregation. Thromboelastographic studies showed that the anticoagulant action of cobra venom is due to the synergistic effects of phospholipase A2 and cardiotoxin.     

毛蚶多糖的分离纯化和免疫活性测定

目的从毛蚶体内分离纯化得到毛蚶多糖精品（polysaccharide from Arca subcrenata Lischke．简称ASLP），分析其纯度和单糖组成，同时考察免疫活性。方法经除蛋白、DEAE-52和Sephadex-GS0柱层析得多糖精品。糖醇乙酸酯法测定单糖组成。体外脾淋巴细胞增殖测定ASLP的免疫活性。结果从毛蚶体内提取到一种均一的多糖组分，其单糖组成只有葡萄糖，ASLP能够显著地促进脾淋巴细胞的增殖。结论从毛蚶中分离纯化得到一种均一的多糖组分，具有显著的体外免疫活性。     

A low-molecular-weight cadmium-binding substance in human and rat livers and human blood

A cadmium-binding substance with a molecular weight even lower than that of metallothionein was demonstrated on Sephadex G-75 gel filtration chromatography of the soluble fractions from newborn human and adult rat liver homogenates and adult human hemolysate which were mixed with CdCl2 in vitro. This substance was purified from rat liver extracts by gel filtration and ion-exchange column chromatography and characterized by 6 M guanidine X HCl thin-layer gel filtration chromatography and N-terminal and total amino acid analyses. The results showed that the isolated low-molecular-weight cadmium-binding substance was a cadmium-reduced glutathione complex, whose molecular weight was found to be approximately 1400 by Sephadex G-15 gel filtration.     

Involvement of tissue sulfhydryls in the formation of a complex of methylmercury with selenium

Albumin-bound methylmercury was converted to a benzene-extractable form by the soluble fraction of rat liver, kidney or brain in the presence of selenite, but not in its absence. The factors in the soluble fraction causing this conversion were investigated by column chromatography. Sephadex G-25 chromatography showed that effective factors were present in non-protein and protein fractions. It was concluded from ion exchange and Sephadex G-200 chromatography that these factors in the non-protein and protein fractions were reduced glutathione (GSH) and protein sulfhydryl groups respectively. Because GSH and the soluble protein could be replaced by sulfhydryl compounds, such as cysteine and 2-mercaptoethanol, as well as by a purified protein with sulfhydryl groups, reduced ribonuclease (RNase), respectively, it was concluded that sulfhydryl groups of GSH and/or proteins in the soluble fraction were needed for selenite-induced conversion of methylmercury to a benzene-soluble form. Among the various selenium compounds tested, only H₂Se (the reduced metabolite of selenite) was found to react directly with methylmercury to form a benzene extractable mercury compound in the absence of the soluble fraction. These findings suggest that the conversion of methylmercury to a benzene-soluble form occurs by reaction of methylmercury with selenium (possibly H₂Se) reduced by GSH and/or protein sulfhydryl groups in the soluble fraction. Thin-layer chromatography showed that benzene-extractable mercury consists mainly of bis(methylmercuric) selenide (BMS). A minor component, trismethylmercuric selenonium, was also detected by mass spectrography.     

DEMONSTRATION OF AN ENDOGENOUS,HIGHLY POTENT,NONCOMPETITIVE PROTEIN INHIBITOR(S) OF 3H-DIAZEPAM BINDING IN BOVINE BRAIN

Extracts from bovine brain show significant diazepam displacing activity in receptor binding assay. The proteinase sensitive component of this benzo-diazepine-like activity has been resolved into three main fractions by chromatography on a Sephadex G-75 column. The most active fraction that contains the largest proteins has been further characterized and shown to be a noncompetitive inhibitor of the diazepam binding to synaptic brain membranes.     

中华眼镜蛇毒核糖核酸酶的分离纯化及其特殊生物学活性

目的从中华眼镜蛇毒中分离纯化核糖核酸酶,并研究其生物学活性。方法以中华眼镜蛇毒为原料,采用SP-Trisacryl阳离子交换色谱、Sephadex G-75凝胶色谱、C8反相色谱等纯化方法,分离纯化具有核糖核酸酶活性的蛋白质,表征其酶学性质,检测其抑菌性、抗肿瘤细胞作用及抗氧化作用。结果 SDS-PAGE电泳显示纯化的中华眼镜蛇毒核糖核酸酶(Na-RNase)为相对分子质量为13 000的单一成分。该酶最适反应温度为40℃,最适pH值为6.5,米氏常数为3.67μmol/L,最大反应速率为3.52 pmol/s。在体外抑菌实验中,在8μmol/L的浓度下对大肠艾希菌和金黄葡萄球菌均未显示出显著抑制作用;在体外细胞毒性实验中,对于Hela肿瘤细胞和正常的人成纤维细胞在8μmol/L的浓度下无明显抑制作用;在抗氧化实验中,浓度达到71.5μmol/mL时,对小鼠肝微粒体脂质过氧化反应的抑制率为41.30%。结论中华眼镜蛇毒中的核糖核酸酶具有抗氧化作用,可能作为氧化还原的调节剂,在有关的疾病治疗方面具有应用的潜能。