具有谷胱甘肽过氧化物酶活性的含硒人源单链抗体的制备

以谷胱甘肽(GSH)为靶抗原, 从噬菌体展示人源单链抗体库中筛选人源单链抗体(scFv). 经3轮筛选后, 用ELISA方法检测出5个(2, 11, 16, 24, 32 )可以和GSH结合的克隆. PCR产物的电泳和测序结果表明, 只有3个克隆(11, 16, 24)具有完整的scFv编码基因. 选取和GSH结合力高的克隆11的scFv 编码基因组装到表达载体pPELB上, 在大肠杆菌Rosetta中进行可溶性表达, 用Ni2+螯合亲和层析纯化scFv-11, 免疫点印迹结果证实该抗体能与GSH特异结合. 通过化学突变将scFv-11的丝氨酸转变成硒代半胱氨酸(Sec)后, 获得了具有谷胱甘肽过氧化物酶(GPX)活力的含硒(Se)人源单链抗体(Se-scFv-11), 其活力为351 U/μmol.     

基于GSH-CdTe/CdS量子点的荧光变化研究hsDNA与盐酸洛美沙星-Cu(Ⅱ)配合物的相互作用

采用水相法合成了谷胱甘肽(GSH)修饰的CdTe/CdS量子点(GSH-CdTe/CdS QDs). 透射电子显微镜表征结果表明, GSH-CdTe/CdS QDs的粒径分布均匀, 分散性好. 在Tris-HCl(pH=7.6)缓冲液中, 由于静电引力作用, 带正电的盐酸洛美沙星(LMFH)-Cu(Ⅱ)配合物[LMFH-Cu(Ⅱ)]吸附到带负电的GSH-CdTe/CdS QDs表面形成基态复合物, 导致GSH-CdTe/CdS QDs的荧光猝灭. 随后, 向GSH-CdTe/CdS QDs-LMFH-Cu(Ⅱ)配合物体系中加入鲱鱼精DNA(hsDNA), hsDNA可诱导LMFH-Cu(Ⅱ)配合物从GSH-CdTe/CdS QDs表面脱落而嵌入到hsDNA的双螺旋结构中, 使GSH-CdTe/CdS QDs的荧光恢复. 通过对GSH-CdTe/CdS QDs荧光的可逆调控, 利用荧光光谱、 紫外-可见吸收光谱和共振瑞利散射光谱研究了hsDNA与LMFH-Cu(Ⅱ)配合物的相互作用. 通过对比GSH-CdTe/CdS QDs与LMFH相互作用的光谱性质, 讨论了GSH-CdTe/CdS QDs-LMFH-Cu(Ⅱ)-hsDNA的相互作用机理, 模拟了作用过程, 从而建立了一种研究氟诺喹酮类药物的金属配合物与核酸相互作用机制的光谱方法.     

溶胶-凝胶分子印迹CsPbBr3量子点的构建及其在胆固醇荧光检测中的应用

该文研究了CsPbBr3量子点（QDs）作为一种新型荧光（FL）探针对胆固醇（CHO）的检测。以3-氨丙基三乙氧基硅烷（APTES）为功能单体,CHO为模板分子,采用溶胶－凝胶技术,将CHO引入到SiO2网络中,制备了CHO分子印迹膜保护的CsPbBr3@SiO2 QDs。该分子印迹膜既可降低空气、湿度等与CsPbBr3 QDs的接触,提高CsPbBr3 QDs的稳定性,又赋予CsPbBr3 QDs对CHO的选择性。检测过程中,CHO被捕获到CsPbBr3@SiO2QDs的印迹位点上,形成空间位阻,导致CsPbBr3 QDs荧光强度下降。在最优条件下,CsPbBr3 QDs的荧光猝灭效率和CHO浓度对数在1.00 × 10－11 ~ 5.00 × 10-8 mol·L－1范围内呈现良好的线性关系,检出限为2.48 × 10－12 mol·L－1。结果表明,该方法灵敏度高、选择性好。该工作为CHO检测提供了一种新的策略,拓宽了CsPbBr3 QDs的应用范围。     

利用水相合成的量子点标记木瓜蛋白酶的研究

利用半导体纳米粒子 (也称半导体量子点 ,Quantum Dots,以下简称 QDs)和表面修饰技术制备的半导体荧光探针具有极其优良的光谱特征和光化学稳定性 [1] .自 1 997年以来 ,随着量子点制备技术的不断提高 ,量子点在生物医学方面已有应用 . 1 998年 ,Alivisatos[1] 和 Nie[2 ] 两个研究小组分别将结合了生物分子的 QDs作为荧光探针应用于生物体系 ,开创了纳米粒子应用的新领域 .最近 Nie等 [3 ]在利用量子点编码生物分子的研究中取得了突破性进展 .目前 ,纳米粒子与生物分子的连接以共价键方式相结合最为常见 [1,2 ,4 ,5] ,而且在这些应用中…     

量子点标记天花粉蛋白的研究

将半导体量子点应用于记天花粉蛋白,研究了量子占标记天花粉蛋白的吸收光谱、荧光光谱和酶活性变化,与游离的QDs 相比,QDs-TCS的吸收光谱在400－600nm范围内不发生明显变化,QDs-TCS的发射光普发生蓝移,但发射半宽不变,标记上QDs后TCS的酶活性没有发生改变,利用双光子激发扫描荧光显微镜和激光共取焦显微镜同时观察QDs-TCS在人绒癌细胞内的分布发现,QDs-TCS能够跨膜进入细胞,并在细胸核周围呈聚集分布。     

QDs标记免疫调节肽及其与T细胞作用的表征

量子点是直径为1~10 nm的球形半导体纳米晶体, 也被称为半导体量子点, 简称QDs. 与有机荧光染料相比, QDs具有激发光谱单一、 荧光谱线窄、 发光效率高、 发光颜色可调、 可进行多色联合标记, 并且光稳定性好等优点, 所以量子点是非常有前途的生物标记物[^1,2]. 研究结果表明, 量子点可以与许多生物分子如蛋白质、多肽、核酸及小分子配体等偶联. 现已有许多关于量子点标记生物分子的报道, 如用量子点标记木瓜蛋白酶、 胰蛋白酶、 天花粉蛋白和表皮生长因子等^[3-5].用量子点标记生物分子作为荧光探针已成功地应用于多种生物分析, 如DNA杂交监测、 免疫分析和用QDs检测ATP推动的反应等^[4,6,7]. 目前, 对量子点标记生物分子的报道多为对大分子蛋白质的标记, 而对小分子肽标记的报道却很少.     

CdTe量子点与L-天冬氨酸相互作用的研究及其分析应用

水相中合成了谷胱甘肽(GSH)修饰的CdTe量子点(GSH-CdTe QDs). 利用紫外-可见吸收光谱(UV-vis)、共振瑞利散射(RRS)光谱、荧光光谱(FL)和傅里叶变换红外光谱(FTIR)研究了GSH-CdTe QDs与L-天冬氨酸(L-Aspartic acid, L-Asp)的相互作用机理. 在pH为5.3 的BR缓冲溶液中, GSH-CdTe QDs与L-Asp主要通过静电引力和氢键作用在GSH-CdTe QDs周围形成了粒径较大的聚集体, 导致体系的RRS显著增强和GSH-CdTe QDs的荧光急剧猝灭. 在一定浓度范围内RRS增强和荧光猝灭均与L-Asp的浓度成正比, 其中RRS法灵敏度更高（对L-Asp检出限为14.2 ng•mL^–1）, 据此建立了以GSH-CdTe QDs作RRS探针测定L-Asp的一种新方法. 同时讨论了反应条件和共存物质对体系RRS强度的影响. 用于实际尿样中L-Asp的测定, 实验结果满意.     

Mn掺杂ZnS量子点荧光猝灭法测定对乙酰氨基酚

利用水热合成法合成了L-谷胱甘肽(GSH)修饰的Mn掺杂ZnS量子点(QDs),基于对乙酰氨基酚对该量子点的荧光猝灭作用,建立了一种快速高效检测对乙酰氨基酚的方法.在最优实验条件下,量子点荧光强度的变化与对乙酰氨基酚浓度在0.25~10μmol·L-1范围内呈良好的线性关系(R=0.999 5),检出限为4.1×10-8mol·L-1,相对标准偏差为0.35%.该方法成功地应用于人尿液中对乙酰氨基酚的测定,样品加标回收率为95%~100%.     

水溶性量子点的制备及其与壳聚糖衍生物的自组装

以3-巯基丙酸(HS-CH2CH2COOH)为稳定剂, 制备了水溶性的碲化镉(CdTe)量子点(QDs), 考察了制备条件对QDs荧光性能的影响及CdTe QDs与壳聚糖及叶酸和聚乙二醇改性的壳聚糖的自组装. 研究发现, 壳聚糖及改性壳聚糖与QDs的复合物荧光强度相对纯的CdTe QDs明显增强, 且QDs被包裹在内核, 复合粒子呈明显的核/壳结构. 改性壳聚糖/QDs复合物较小且尺寸分布更为均一.     

荧光可逆调控研究CdTe量子点-吖啶橙-小牛胸腺DNA的相互作用及分析应用

水相合成了谷胱甘肽(GSH)修饰的CdTe 量子点(QDs). 在PH＝7.4的Tris-HCl缓冲溶液中, 吖啶橙(AO)通过静电引力吸附到GSH-CdTe QDs 的表面, 与GSH-CdTe QDs形成了基态复合物, 导致GSH-CdTe QDs的荧光猝灭. 在GSH-CdTe QDs-AO体系中加入小牛胸腺DNA (ctDNA), ctDNA诱导AO从GSH-CdTe QDs表面脱落嵌入其双螺旋结构中, 导致GSH-CdTe QDs的荧光恢复. 根据GSH-CdTe QDs荧光的猝灭和恢复, 实现了量子点荧光的可逆调控. ctDNA引起GSH-CdTe QDs-AO体系荧光恢复强度与ctDNA浓度成良好的线性关系, 检出限为0.13 ng•mL^－1, 据此提出了简便快捷、准确、高灵敏测定ctDNA的新方法. 还结合共振瑞利散射(RRS)光谱、吸收光谱和原子力显微镜照片研究了GSH-CdTe QDs-AO-ctDNA三者之间的相互作用, 对相互作用机理进行了讨论并提出了相应的作用模型.     

Rapid Selection of Phage Se-scFv with GPX Activity via Combination of Phage Display Antibody Library with Chemical Modification

IntroductionReactive oxygen species(ROS) are known to de-stroy biomacromolecules and cause cell injury[1]. Un-der normal circumstances, there is a balance betweenthe production of ROS and their destruction. Many dis-eases, such as brain ischemia, tumor, v…     

Novel Selenium-containing Human Single-chain Variable Fragment with Glutathione Peroxidase Activity from Computer-aided Molecular Design

In order to enhance the glutathione peroxidase(GPX) catalytic activity of the selenium-containing single-chain variable fragments(Se-scFv), a novel human scFv was designed on the basis of the structure of human antibody and optimized via bioinformatics methods such as homologous sequence analysis, three-dimensional(3D) model building, binding-site analysis and docking. The DNA sequence of the new human scFv was synthesized and cloned into the expression vector pET22b(+), then the scFv protein was expressed in soluble form in Escherichia coli BL21(DE3) and purified by Ni2+-immobilized metal affinity chromatography(IMAC). The serine residue of scFv in the active site was converted into selenocysteine(Sec) with the chemical modification method, thus, the human Se-scFv with GPX activity was obtained. The GPX activity of the Se-scFv protein was characterized. Compared with other Se-scFv, the new human Se-scFv showed similar efficiency for catalyzing the reduction of hydrogen peroxide by glutathione. It exhibited pH and temperature dependent catalytic activity and a typical ping-pong kinetic mecha- nism.     

Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPα.     

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10⁵ Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K _d) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K _d value of 10⁻⁷ M.     

Screening of scFvs against cTnI from Phage Display Antibody Library and Their Expression in E.coli Rosetta

Introduction CardiactroponinI(cTnI),aspecificproteinof cardiacmusclecells,showsa40%dissimilarity withskeletaltroponinI(sTnI)inaminoacidse- quence.Moreover,humancardiacTnIhas31addi- tionalresiduesonitsN-terminalend,whichare notpresentinskeletalforms,thusprovidingahigh potentialforobtainingcardiac-specificantibod- ies[1,2].Themolecularweightofthisproteinis29 kDaandtherefore,itwillbereleasedreasonably rapidlyafteracutemyocardialinfarction(AMI). CTnIoftenappearsinbloodwithinafewhoursaf- ter…     

亲和柱色谱原位酶切法纯化重金属镉结合的金属硫蛋白-3

按照人金属硫蛋白-3(hMT-3)的基因序列,选用大肠杆菌偏爱的密码子合成了全长hMT-3基因,并将其插入大肠杆菌融合表达质粒pALEX的多克隆位点中,在谷胱甘肽-硫-转移酶(GST)下游与GST融合表达。通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导在大肠杆菌表达菌株BL21（DE3）LysS中表达了与重金属离子镉结合的融合蛋白GST-Cd2+-hMT-3。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明融合蛋白主要在超声上清液中。分别通过“先纯化、后酶切”和“亲和柱色谱原位酶切”两种方法纯化了Cd2+-hMT-3,比较了两种方法的纯化效率和得率,表明原位酶切法操作简便,较之“先纯化、后酶切”法减少了洗脱、透析、冻干等步骤,从而也减少了样品的损失,提高了样品的纯度和得率。从摇瓶培养菌液中纯化获得了结合有Cd2+的完整的人金属硫蛋白-3,得率为1.8%。氨基酸组成分析结果表明所获得的Cd2+-hMT-3不含芳香族氨基酸和组氨酸,符合金属硫蛋白的特征;直读电感耦合等离子体发射光谱分析其硫镉原子比为21∶(7.5±0.1),与理论值21∶7基本吻合。     

口蹄疫病毒3ABC基因截短体在毕赤酵母中的表达及鉴定

将长为525 bp的口蹄疫病毒3ABC基因截短体克隆到毕赤酵母表达载体pPIC9K中, 构建了重组表达质粒pPIC9K-3ABCt. 用BglⅡ线性化后, 电转化毕赤酵母菌GS115, 经表型筛选, PCR鉴定, 获得阳性重组菌(GS115/pPIC9K-3ABCt). 然后进行诱导表达, 通过SDS-PAGE和Western blot鉴定表达产物. 结果表明, 重组菌株成功分泌表达了分子量为40000, 具有免疫反应活性, 且呈二聚体形式的目的蛋白. 在96 h时表达量达到最高峰, 占分泌总蛋白的18%, 达到23.4 mg/L. 为进一步研制口蹄疫免疫和感染动物鉴别诊断试剂奠定了基础.     

Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display

Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. “Phage display” enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.