应用XTT法检测白细胞介素—Ⅱ的生物学活性

应用XTT比色法检测IL－2的生物学活性并与MTT法和^3H掺入法进行比较,结果XTT法较上述方法简便易行,结果稳定,重复性好。     

HIV—lgag／IFNα—2b表达产物的生物活性研究

目的 :将艾滋病病毒核心蛋白 (gag)与干扰素 (IFNα - 2b)融合基因表达的融合蛋白作为免疫原免疫小鼠 ,动态观察小鼠的体液免疫、细胞免疫与CTL应答。方法 :将IFNα - 2b基因片段插入到gag基因的nt5 31位点 ,经脂质体转染与血凝素阴性蚀斑筛选 ,挑出重组痘苗病毒。经SDS -PAGE和Westernblot鉴定表达产物。以小鼠为实验对象 ,用重组痘苗病毒vJ38gag/IFNα - 2b免疫小鼠 ,用ELISA方法检测血清IgG抗体含量。用流式细胞仪测定小鼠外周血CD 4 、CD 8T淋巴细胞计数。 3H -TdR掺入法检测细胞毒性T淋巴细胞杀伤活性。结果 :血清IgG抗体含量逐渐增高 ,实验组与对照组比较差异有显著性意义 (p <0 .0 5 )。CD 4 、CD 8T淋巴细胞计数、CTL检测实验组与对照组比较差异均有显著性意义 (p <0 .0 5 )。结论 :重组痘苗病毒vJ38gag/IFNα - 2b能增强小鼠的体液免疫、细胞免疫和CTL应答。IFNα - 2b可以作为免疫佐剂增强机体的免疫状态。     

重组人白介素—12在银纹夜蛾中的表达纯化及其生物活性

KB细胞经PDBu刺激 ,采用异硫氰酸胍一步法提取细胞总RNA ,RT PCR法获得重组人白介素 12 (rhIL 12 )P35和P4 0cDNA。将hIL 12P35cDNA和P4 0cDNA分别克隆到 pAcUW 5 1载体的Polyhedrin和P10启动子下游 ,构建 pAcUW 5 1 IL12转移载体。pAcUW 5 1 IL12与坏死缺陷型线性苜蓿银纹夜蛾核型多角体病毒基因DNA共转染Sf9细胞 ,获得重组杆状病毒Ac hIL12。该病毒经血腔感染银纹夜蛾幼虫 ,采用亲和层析法纯化rhIL 12 ;SDS PAGE(银染法 )、Westernblot鉴定表达和纯化的产物 ;ELISA检测rhIL 12含量 ;MTT法检测rhIL 12样品生物活性。rhIL 12分子量为 75kD。rhIL 12在Sf9细胞培养中表达水平为 17.8μg/10 6细胞 ;在银纹夜蛾幼虫中表达水平为 2 0 0 - 30 0mg/L血淋巴。纯化的重组rhIL 12样品对经PHA P激活的PBMC有明显的增殖活性 ,且具有明显的促NK细胞杀伤活性的生物活性     

MTT实验检测重组人角质细胞生长因子-2诱导不同细胞系增殖的差异

目的:用MTT法检测重组人角质细胞生长因子-2(rhKGF-2)诱导不同细胞系增殖的差异,建立验证rhKGF-2生物活性的细胞模型.方法:利用载体pBV220-KGF-2原核表达和纯化rhKGF-2,然后用MTT法分别检测rhKGF-2诱导大鼠气管上皮细胞RTE、大鼠小肠上皮细胞IEC-6、小鼠成纤维细胞NIH/3T3...     

SelS高表达保护人脐静脉内皮细胞免于H2O2诱导的细胞损伤

采用以下方法探讨SelS在内皮细胞中的表达和作用:将SelS基因克隆到真核表达载体pLNCX2,RT-PCR、XhoⅠ/ClaⅠ双酶切以及DNA序列分析验证目的基因;利用脂质体转染技术将pLNCX2-SelS或pLNCX2转染至人脐静脉内皮细胞(ECV304细胞),RT-PCR检测重组基因SelS的表达;MTT方法检测转染后过氧化氢(H2O2)对内皮细胞增殖能力的影响;硫代巴比妥酸法测定暴露于H2O2中不同转染组细胞脂质过氧化产物丙二醛含量.结果表明:成功构建真核表达载体pLNCX2-SelS;转染后重组SelS mRNA表达水平是内源性水平的1.76倍;H2O2对ECV304细胞损伤后,高表达SelS组细胞活性增强、H2O2诱导产生的丙二醛减少.上述结果表明,高表达SelS可保护内皮细胞免于H2O2诱导的细胞损伤,其作用机制与抗氧化有关.     

Zn掺杂TiO_2薄膜制备及其光催化降解亚甲基蓝

采用溶胶-凝胶浸渍提拉法在普通载玻片上制备了Zn掺杂TiO2薄膜,利用XRD、SEM对薄膜的结构、形貌进行了分析,并通过对亚甲基蓝(MB)溶液的降解率来评价其光催化活性。XRD结果表明:当掺入的锌含量达到1wt%时,除了锐钛矿型的TiO2,还出现了一种新相ZnTiO3的衍射峰,其结构为钙钛矿型,通过薄膜的SEM图像也能观察到ZnTiO3。通过对亚甲基蓝降解率的分析发现,掺锌量为1wt%的TiO2薄膜光催化活性较高,光照40min后对MB溶液的降解率高达98.7%。     

大鼠脑线粒体体外蛋白翻译体系的建立及蛋白产物的鉴定

目的 :建立大鼠脑组织线粒体的体外蛋白合成体系并对其合成产物进行电泳分离和分子量鉴定。方法 :分离大鼠脑组织线粒体 ,用3 H 亮氨酸掺入法探索线粒体体外翻译的最佳条件 ,3 5S 蛋氨酸掺入并对翻译后产物经SDS 聚丙烯酰胺凝胶电泳和放射自显影进行分子量鉴定。结果 :分离的线粒体氧化磷酸化偶联程度高 ,呼吸控制率(RCR)在 3.5～ 5 .5之间 ;体外3 H 亮氨酸的掺入活性在 6 0min内近似线性增长 ,而后维持在一相对稳定水平 ;3 H 亮氨酸的掺入活性随线粒体蛋白浓度而增加 ,而单位线粒体蛋白的掺入活性在 1mg/ml时最高 ;3 5S 蛋氨酸掺入SDS 聚丙烯酰胺凝胶电泳后可观察到清晰的 8条自显影带 ,分子量分别为 (单位Kda) 86、6 6、5 6、43、33、2 9、2 5、18。结论 :用此方法建立的脑线粒体离体翻译反应体系具有高活性和翻译忠实性等特点 ,是研究脑mtDNA在翻译水平的表达及调控的有效方法     

突变型人白细胞介素-2基因在巴斯德毕赤酵母中的表达

为了提高人重组白细胞介素 2的稳定性和活性以及减少毒副作用 ,有必要定向改造rhIL 2的分子结构 .用PCR法从白细胞介素 2 (IL 2 )cDNA全序列中扩增成熟的肽基因片段 ,并利用定点突变技术将人重组白细胞介素 2第 12 5位游离的半胱氨酸编码序列突变为丙氨酸序列 .编码 18位亮氨酸的序列突变为蛋氨酸序列 ;编码 19位亮氨酸的序列突变为丝氨酸序列 .突变型人白细胞介素 2 (MvIL 2 )基因与表达载体pPIC9K重组 ,酶切线性化后用Invitrogen转化毕赤酵母试剂盒导入酵母细胞进行整合 ,经筛选得到一高表达白介素 2的克隆 .SDS PAGE显示 ,表达量约占总量的4 5 7% .经Western印迹验证 ,重组人白介素 2有免疫活性 ;与野生型IL 2相比 ,所获得的突变型IL 2纯品的比活性为 4 0× 10 7IU mg蛋白 ,比天然型IL 2高 4～ 5倍     

重组MBP-SUV39H1在大肠杆菌中表达与纯化

[目的]在大肠杆菌中获得具有甲基转移酶活性的重组MBP-SUV39H1蛋白。[方法]通过在大肠杆菌中同时表达异染色质蛋白1(HP1)与重组MBP-SUV39H1蛋白的方法,实现了MBP-SUV39H1的表达,采用his亲和纯化与分子筛Superdex200(SD200)两步分离纯化方案,并利用质谱和ELISA检测MBP-SUV39H1的甲基转移酶活性。[结果]利用大肠杆菌成功表达了MBP-SUV39H1融合蛋白,经纯化后目的条带单一,并具有良好的甲基转移酶活性。[结论]纯化的具有甲基转移酶活性的MBP-SUV39H1可用于抑制剂筛选等后续研究。     

以Hsp90为靶点的4-(4-羟基-3-甲氧基苯亚甲基)姜黄素抗肿瘤活性研究

合成4-(4-羟基-3-甲氧基苯亚甲基)姜黄素(C085),研究其体外抗肿瘤活性和抑制Hsp90作用。以香草醛和姜黄素为原料,微波条件下Knovenagel缩合反应合成了4-(4-羟基-3-甲氧基苯亚甲基)姜黄素(C085)。以姜黄素为对照,MTT法考察目标化合物对人乳腺癌细胞SKBr3、人慢性粒细胞白血病急变细胞株K562、人急性髓系白血病细胞株HL-60、人肝肿瘤细胞株Hep G2、小鼠黑色素瘤细胞株B-16、人结肠癌细胞株SW480、人胰腺癌细胞株Bxpc-3、人神经母细胞瘤细胞株SH-SY5Y、人胃癌细胞株MGC80-3的抑制活性。对上述细胞株的IC50值依次为0.51、1.26、2.90、0.81、1.77、1.31、8.22、1.93、7.41μmol/L,对多种肿瘤细胞抑制活性明显强于Cur。Western Blot结果表明C085明显下调Hsp90的客户蛋白Her2和AKT的表达水平。分子对接分析也支持C085是Hsp90抑制剂。     

Detection of double-stranded RNA-protein interactions by methylene blue-mediated photo-crosslinking.

Double-stranded(ds) RNA-binding proteins have diverse functions in the cell. An obstacle to investigating the interactions between these proteins and dsRNA is the relative inefficiency of traditional UV-crosslinking methods for extended regions of dsRNA. We have therefore developed an alternative procedure for RNA-protein photo-crosslinking that efficiently induces RNA-protein crosslinks in double-stranded regions of RNA. We show that dsRNA-protein crosslinks can be induced by visible light in the presence of the dye methylene blue, which most likely mediates crosslinking by intercalating in the dsRNA helix. A recombinant dsRNA binding domain from the Drosophila staufen protein and human protein kinase R were crosslinked by UV or methylene blue to a series of dsRNAs. In each case, the degree of crosslinking was greater with methylene blue, particularly with RNAs with few single-stranded loops. Methylene blue-mediated crosslinking therefore complements and extends the existing repertoire of crosslinking methods for detecting RNA-protein interactions.     

Acid-labile sulfide and zero-valence sulfur in plant extracts containing chlorophyll and ionic detergents

Two methods for analysis of acid-labile sulfide and zero-valence sulfur in plant extracts containing chlorophyll as well as ionic and/or nonionic detergents are presented. Both methods are based on the conversion of sulfide into methylene blue. In the first method an ethyl acetate extraction step is used to remove chlorophyll and its degradation products which otherwise prevent spectrophotometric quantitation of methylene blue. The second assay method employs 35S-labeled plant extracts. This method, which involves thin-layer chromatography and autoradiography, is potentially more sensitive than the spectrophotometric assay in detecting acid-labile sulfide and zero-valence sulfur.     

人白细胞介素11的定点聚乙二醇修饰

利用人白细胞介素11（hIL-11）无半胱氨酸（Cys）残基这一特点,通过定点突变将一个Cys残基引入hIL-11的N末端。然后,利用与Cys 巯基特异性反应的mPEG-马来酰亚胺将mPEG偶联到预先选定的位点,经层析纯化得到hIL-11的定点PEG修饰物。利用依赖型细胞株7TD1测定其生物学活性,结果表明,其体外生物学活性保持原有hIL-11活性的30%左右。定点聚乙二醇修饰方法为定向改造hIL-11,提高其药效的应用研究打下基础。     

Analysis of methylene blue in human urine by capillary electrophoresis

A capillary electrophoresis method for the determination of the dye methylene blue (tetramethylthionine, MB) in human urine depending on liquid/liquid-extraction and diode array detection has been developed, validated, and applied to samples of healthy individuals, who had been dosed with methylene blue within clinical studies. After extraction with dichloromethane and sodium hexanesulfonate, sample extracts were measured on an extended light path capillary. The dye was detected simultaneously at 292 and 592 nm using methylene violet 3 RAX as internal standard. The limit of quantification was 1.0 microg/ml. The accuracy of the method varied between -15.2 and +0.8% and the precision ranged from 2.0 to 12.0%. The method was linear at least within 1.0 and 60 microg/ml. In contrast to earlier indirect determinations no leuco methylene blue (LMB) was directly detected in urine, whereas in aqueous test solutions containing surplus amounts of ascorbic acid leuco methylene blue was well separated from MB in a single run.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

酿酒酵母菌耐热性快速鉴别的方法研究

目的:找出快速有效的鉴别酵母耐热性的方法。方法:酿酒酵母菌经过不同温度热激处理后,用美兰染色计数、稀释平板计数、发酵活力直接测定法和浊度测定法对酿酒酵母菌耐热性进行了测定,同时对酵母菌的形态也进行了观察。结果:表明浊度测定的结果和发酵活力直接测定法测定的结果关系没有相关性,不便用来测定酵母菌的活力。用美兰染色计数、稀释平板计数和发酵活力直接测定法所得的结果有同样的趋势,通过相关性分析,这些方法都可以表示酵母菌的活力,但各有其特点。其中,美兰染色计数是鉴别酵母耐热性的快速有效的方法。     

Growth stimulation produced by methylene blue treatment in sweet potato

Methylene blue as an alternative treatment to gamma rays to stimulate growth in sweet potato tissue cultures, was applied in two different ways: – pre-incubation of nodal explants with methylene blue for 1 h using urea as a permeabilizer; – methylene blue directly incorporated into the culture medium. Both treatments stimulated growth, but the better performance being obtained with the second treatment, which had no toxic effect. The activity and electrophoresis pattern of peroxidase after treatment ofIpomoea batatas plantlets with methylene blue or gamma rays did not show similar results for the two treatments. Peroxidase activity was greater in leaves of gamma ray treated plants compared to the non-treated control. The results obtained with the Methylene blue treatment did not significantly change the peroxidase activity relative to the control. This revised version was published online in June 2006 with corrections to the Cover Date.     

人白介素-4与大肠杆菌二硫键异构酶DsbC在大肠杆菌中的融合表达及羟胺切割后共复性

目的:通过融合表达、羟胺切割、与二硫键异构酶共复性,获得高表达、高纯度、高生物活性的重组人白细胞介素-4(rhIL-4)。方法:将5端引入了羟胺切割位点的hIL-4基因克隆到大肠杆菌二硫键异构酶DsbC的原核表达载体pET-DsbC中,IPTG诱导表达,对包涵体进行纯化,然后在变性条件下经羟胺切割,利用DsbC的分子伴侣功能与hIL-4进行共复性,最后利用阳离子交换层析纯化获得rhIL-4蛋白。结果:融合蛋白DsbC-hIL-4的表达量占细菌总蛋白的40%以上,以包涵体形式存在;纯化后得到的rhIL-4的相对分子量为15×103,与预期一致,电泳纯度达95%;细胞学实验测定其具有良好的生物学活性。结论:通过融合表达的方法可以提高hIL-4的原核表达量;利用共复性的方式极大地提高了hIL-4的复性率和生物活性。     

Purification and characterization of recombinant human interleukin 5 expressed in Chinese hamster ovary cells

Recombinant human interleukin 5 (rhIL-5) expressed in Chinese hamster ovary cells was purified and characterized. Molecular heterogeneity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two major components of Mr around 40,000 were detected under non-reducing conditions. However, under reducing conditions, the Mr of rhIL-5 was determined to be 22,000 and 20,000. After treatment with endoglycosidase F, a band with an apparent Mr of 18,000 was observed. Treatment of rhIL-5 with 2-mercaptoethanol followed by N-ethylmaleimide resulted in its dissociation into a monomeric form. This alkylated rhIL-5 was biologically less active than intact rhIL-5. These results suggest that rhIL-5 exists as a dimer, and that the heterogeneity of rhIL-5 is mainly due to the difference in the content of carbohydrate. Moreover, the formation of disulfide bond(s) might be important for the biological activity of rhIL-5.