靶向喉癌的蜂毒肽重组抗体的构建及鉴定

利用大肠杆菌表达系统制备了重组融合蛋白antiEGFR/MEL,并用Ni2+层析柱对其进行了纯化.该重组蛋白中抗表皮生长因子受体(antiEGFR)单链抗体(scFv)主要靶向喉癌细胞中的EGFR,而蜂毒肽(MEL)主要抑制肿瘤细胞增殖.采用SDS-PAGE和Westernblot检测证明了antiEGFR/MEL的有效表达.共聚焦显微镜和流式细胞术实验结果表明,antiEGFR/MEL可与Hep-2肿瘤细胞有效结合,而几乎不与EGFR阴性的Jurkat细胞结合.噻唑蓝(MTT)检测结果说明,antiEGFR/MEL可有效抑制人喉癌细胞Hep-2的增殖.以上结果表明,antiEGFR/MEL能够有效靶向EGFR阳性肿瘤细胞,并有效抑制肿瘤细胞增殖,有望应用于EGFR靶向肿瘤治疗.     

花生、河虾过敏原蛋白的纯化及其抗体的制备和鉴定

本文对花生、河虾中的主要过敏原蛋白进行分离纯化并制备抗体,同时探索胶体金作为免疫佐剂的可能性。结果表明,经电泳检验纯化后的过敏蛋白纯度超过90%,胶体金能够显著提高大白兔体内抗花生、河虾过敏蛋白的水平,花生与胶体金组的平均效价比弗氏佐剂组提高了一倍,其Ic50为34.28 ng/mL;河虾与胶体金组的平均效价比弗氏佐剂组提高了四分之一,其Ic50为43.95 ng/mL。该研究成功获得花生、河虾中主要过敏原组分及其抗体,且胶体金有可能开发为新一代免疫佐剂。     

环境激素邻苯二甲酸二丁酯免疫检测Ⅱ——抗体的制备

邻苯二甲酸二丁酯(DBP)半抗原衍生物通过偶氮键与载体蛋白BSA偶合后作为免疫原,免疫新西兰大白兔,得到合格的DBP抗血清,双向琼脂扩散方法测定血清效价为1∶32～1∶64,间接荧光免疫法检测效价为1∶6 400～1∶12 800。通过兔颈部采血、抗血清纯化,得到的抗邻苯二甲酸二丁酯多克隆抗体IgG冻干后,分装在-20℃低温保存。建立了间接荧光免疫法检测纯化抗体的方法。间接竞争荧光免疫法考察交叉反应试验结果显示,纯化抗邻苯二甲酸二丁酯抗体与邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丙酯、邻苯二甲酸二环己酯、邻苯二甲酸二戊酯、邻苯二甲酸二(2-乙基己基)酯等邻苯二甲酸酯类环境激素的交叉反应均小于10%。制备的抗体可应用于环境激素DBP的荧光免疫分析。     

抗肿瘤双特异免疫导向治疗制剂CAtin的表达及活性分析

结合生物信息学方法与已知癌胚抗原(Carcinoembryonic antigen, CEA)特异性单链抗体(Single chain Fv fragment, scFv)核苷酸序列, 经分子设计和密码子优化后, 通过化学方法合成CEA二硫键稳定性单链抗体(Disulfide stabilized single chain Fv fragments, scdsFv)基因片段. 将凋亡素基因(Apoptin)通过一段柔性连接肽(Linker)连接在CEA scdsFv基因下游, 并克隆入大肠杆菌表达载体质粒pET28a, 转化BL21感受态菌后经异丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside, IPTG)诱导, 表达融合蛋白CAtin. SDS-PAGE和Western-Blot分析表明, 目的蛋白得到良好表达, 经条件优化后表达量最高可达44.1 mg/L. 融合蛋白经分步洗涤法和谷胱甘肽对表达的目的蛋白进行初步纯化和复性后, 利用人肝癌细胞(HCC)对所制备融合蛋白进行亲和力测定、细胞结合活性测定和特异性细胞杀伤活性分析. 结果显示, 所制备融合蛋白不仅能够有效地与上述肿瘤细胞结合, 并对其具有明显的杀伤活性, 表明成功制备了具有特异性识别和特异性杀伤活性的双特异抗肿瘤免疫导向制剂.     

金纳米棒的制备、生长机理及纯化

金纳米棒由于其独特物理性质而在众多的各向异性金纳米颗粒中赢得了关注。目前,金纳米棒在纳米电子学、光学、生物医药等研究领域均具有良好的应用前景。对金纳米棒合成的有效调控直接决定着其形状、尺寸和长径比,而这些又进一步影响着金纳米棒的物理性质。本文梳理了金纳米棒制备方法的发展脉络,以模板法、电化学方法、种子生长法以及近年来出现的无种子生长法为主线,系统综述了金纳米棒制备过程实验参数调控对产物结构、物理性质的影响,详细阐述了关于单晶以及孪晶金纳米棒的生长机理,并介绍了提高产物纯度的分离纯化手段。     

低分子量紫色酸性磷酸酶多克隆抗体的制备及应用

将最近从地瓜中提出来的低分子量紫色酸性磷酸酶（smPAP)基因克隆到GST融合蛋白表达载体pGEX－2T中，在大肠杆菌BL21codon plus中进行表达，用表达的融合蛋白免疫兔子产生多克隆抗体，用抗血清在昆虫细胞中表达的smPAP和地瓜提取液中分离纯化的PAP进行检测，产生了良好的交叉反应。     

抗黄曲霉毒素B1多价纳米抗体融合蛋白的构建及活性分析

纳米抗体来源于天然缺失轻链的重链抗体可变区,是已知最小抗原结合单元.该研究构建了抗黄曲霉毒素B1(AFB1)纳米抗体的单价及多价串联体,分别与绿色荧光蛋白(GFP)编码片段融合并克隆至原核表达载体pET30.以大肠杆菌BL21(DE3)作为表达宿主,通过异丙基-β-D硫代吡喃半乳糖苷诱导,亲和层析技术分别纯化单、双及三...     

SPAP2基因的克隆、融合表达及分离纯化

本文构建的表达载体pGex-2T-SPAP2CT在大肠杆菌中表达出可融性蛋白, 其分子量为46 000, 经纯化后得到产率为10%、 纯度大于90%的GST-SPAP2CT蛋白.     

[Bl0Glu，B24-Asp-B25]人胰岛素类似物的制备、鉴定与性质研究

采用PCR方法，将人胰岛素分子B链B10位His突变为Glu，在B24和B25位之间插入Asp．构建了[B10Glu，B24-Asp-B25]胰岛素原基因、利用通用型质粒pBV220构建表达载体，在大肠杆菌DH5a中表达．表达蛋白为包含体形式．约占菌体总蛋白的20％～30％．经过复性和凝胶过滤得到胰岛素原融合蛋白、用胰蛋白酶和羧肽酶B酶切．经DEAE离子交换和RP-HPLC纯化得胰岛素突变体类似物．用凝胶过滤法测定了蛋白质分子自身的缔合性质．用圆二色谱测定了构象变化、放射性免疫活性及受体结合活性测定结果表明，突变体分子缔合性明显下降．放免活性和受体结合活性分别约为人胰岛素的73．6％和146％。     

低聚壳聚糖降解制备、分离、纯化、鉴别的研究进展

查阅了近十年来国内外有关壳聚糖降解的最新文献,系统地综述了壳聚糖降解制备、分离、纯化、鉴别的研究进展。     

PAUF promotes adhesiveness of pancreatic cancer cells by modulating focal adhesion kinase

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.     

Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

Phosphatase of regenerating liver 3(PRL3),which belongs to the superfamily of protein tyrosine phosphatases(PTPs),represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes.Presented here are the results of cloning,prokaryotic expression,purification,and polyclonal antibody preparation of PRL3.To obtain a specific polyclonal antibody against PRL3,the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns.Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein.The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.     

抗酮洛芬多克隆抗体的制备及其间接竞争ELISA检测方法的建立

分别将酮洛芬与牛血清白蛋白(BSA)及卵清蛋白(OVA)偶联制得免疫原和包被原,经过免疫新西兰白兔制备多克隆抗体,抗体经纯化后效价为1:128000。使用自制的抗体,建立了测定酮洛芬的间接竞争酶联免疫吸附(ic-ELISA)新方法。ic-ELISA的线性范围为0.010～10.0μg/L,IC50为0.235μg/L,最低检测限为0.0040μg/L,线性回归方程为y=-22.97ρ+104.5(R2=0.980),与布洛芬、双氯酚酸的交叉反应率均小于4%,方法可用于水体中酮洛芬的检测。     

Theoretical study of disubstituted pyrrolopyrimidines as focal adhesion kinase inhibitors

An in silico molecular modeling study of selected 7H‐pyrrolo[2,3‐d]pyrimidines with FAK inhibitory activities was performed. Rigid docking of each inhibitor at the FAK catalytic site was employed to obtain the most appropriate starting structures, followed by molecular mechanics‐based energy minimizations associated with molecular dynamics at the FAK binding site using the AMBER force field. Theoretical values of interaction energies obtained from the geometry optimization calculations for the protein‐inhibitor complexes were compared with published IC₅₀ values for FAK and showed a reasonable correlation. Based on these results and in view of the geometry of the most potent inhibitors, two new molecular structures were designed as possible FAK inhibitors and submitted to the same theoretical procedures. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Design and evaluation of a real-time activity probe for focal adhesion kinase

Focal adhesion kinase (FAK) has been identified as a potential therapeutic target for the treatment of metastatic cancers. Herein we describe the design, synthesis and optimization of a direct activity sensor for FAK and its application to screening FAK inhibitors. We find that the position of the sensing moiety, a phosphorylation-sensitive sulfonamido-oxine fluorophore, can dramatically influence the performance of peptide sensors for FAK. Real-time fluorescence activity assays using an optimized sensor construct, termed FAKtide-S2, are highly reproducible (Z' = 0.91) and are capable of detecting as little as 1 nM recombinant FAK. Utilizing this robust assay format, we define conditions for the screening of FAK inhibitors and demonstrate the utility of this platform using a set of well-characterized small molecule kinase inhibitors. Additionally, we provide the selectivity profile of FAKtide-S2 among a panel of closely related enzymes, identifying conditions for selectively monitoring FAK activity in the presence of off-target enzymes. In the long term, the chemosensor platform described in this work can be used to identify novel FAK inhibitor scaffolds and potentially assess the efficacy of FAK inhibitors in disease models.     

Fast purification of trace vitellogenin from Chinese rare minnow using protein A-immobilized antibody

Vitellogenin (Vtg) is a highly responsive biomarker for environmental exposure to various estrogenically active compounds. Here we present a simple, fast, mild, and stable immobilization of anti-Vtg antibody, and demonstrate its powerful applications for preconcentration and purification of fish Vtg proteins, allowing for the monitoring and screening of environmental exposure to estrogenically active compounds. In this immobilization method, rabbit antiserum containing a specific polyclonal antibody against Vtg was directly immobilized on an antibody-binding Staphylococcal protein A matrix (SpA) without the need for prior purification. Under the unique elution conditions, the Vtg protein can be eluted out alone without any leaked specific antibody. The developed method was further used to purify Vtg from whole-body homogenate of Chinese rare minnow. Compared with previous purification methods, the isolated Vtg fraction by this method displays higher purity and well-preserved structure integrity. Moreover, our method is eight times faster. The simple one-step protein A-based specific antibody immobilization and its associated elution strategy may be extended to a number of antibodies for various application purposes, highlighting the paramount advantages over traditional immunoprecipitation and covalent immobilization of antibodies, and suggesting a wide range of promising applications in environmental monitoring and proteome analysis.     

Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-β-alanine-ovalbumin (TNP-β-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-β-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 × 10⁻¹¹ to 3 × 10⁻⁷ g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 × 10⁻¹¹ g/ml.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.