铜绿微囊藻生物钟蛋白的表达纯化与抗体制备

为了获得融合表达的铜绿微囊藻(Microcystic aeruginosa)生物钟蛋白KaiA、KaiB、KaiC并制备其相应的多克隆抗体,将kaiA、kaiB、kaiC基因分别克隆到原核表达质粒pET-His中.重组质粒pET-His-KaiA,pET-His-KaiB和pET-His-KaiC经酶切和测序鉴定后,分别转化E.coli BL21(DE3)进行融合表达.经SDS-PAGE分析可知,融合表达的KaiA、KaiB和KaiC蛋白表达量可分别达到菌体总蛋白的25%、40%和20%.经亲和层析后融合蛋白KaiA和KaiB的纯度分别达95%和92%,而KaiC经胶回收纯化后纯度也可达93%.将纯化后的三种Kai蛋白作为抗原分别免疫小鼠制备多克隆抗体,经ELISA检测抗体滴度表明,制备的抗KaiA、抗KaiB和抗KaiC的多克隆抗体效价高,分别可达到1:50000、1:60000和1:100000.Western blotting结果表明:获得的多克隆抗体具有较高的效价,抗体能识别相应的Kai蛋白,具有较高的特异性,能用于铜绿微囊藻生物钟蛋白KaiA、KaiB和KaiC的表达节律检测.     

噬菌体显示技术及其应用

噬菌体显示技术的原理在于将外源性基因与噬菌体本身的壳蛋白Ⅲ基因５’端相连，随后利用外源性插入片段与壳蛋白基因表达的融合蛋白位于噬菌体表面的优点，使目的噬菌体的筛选，富集更为简便，有效，在此基础上发展起来的噬菌体抗体，噬菌体抗体库以有机多肽库技术则为抗体的抗备，配体的发现，药物的设计与筛选提供了有效的手段。     

HIV-1核心蛋白的表达与纯化

将编码人I型免疫缺陷病毒(HIV1)核心蛋白p24gag的基因序列克隆到原核表达载体pET28(b)中,高效表达了N,C端融合His·Tagp24蛋白,所表达的重组p24蛋白占菌体总蛋白的46%。在变性条件下,使用NiNTA亲和层析法纯化了p24蛋白,纯度为94%。菌体中及纯化、复性后的目的蛋白均能与抗HIV1p24单克隆抗体发生特异性反应。用纯化的p24蛋白免疫小鼠,4周时小鼠血清抗p24抗体效价达1∶400。实验结果表明:大肠杆菌表达的HIV1p24蛋白纯化后可用作HIV1检测试剂的原料。     

应用全合成噬菌体抗体展示库技术实现抗膀胱癌单链抗体的定向进化

单链抗体在肿瘤治疗的临床应用中常会遇到两方面的问题，即低亲和力和鼠源性导致的免疫原性。为此，我们对鼠源抗膀胱癌单链抗体进行人源化改造，并从噬菌体抗体库中筛选具有高亲和力的人源化的单链抗体分子。运用噬菌体展示技术，成功构建了库容为10^6人源化的噬菌体抗体库，用膀胱癌细胞系膜抗原进行三轮筛选，得到了2个结合抗原活性高于起始的鼠源单链抗体的人源化抗膀胱癌单链抗体。     

中国明对虾蜕皮抑制激素在大肠杆菌中的表达与分离纯化

采用大肠杆菌表达系统对中国明对虾蜕皮抑制激素(MIH)进行了体外重组表达研究.重组蛋白以包涵体的形式存在于菌体中.尿素-SDS-PAGE分析表明,经1mmol/L的IPTG诱导4h后,重组蛋白获得了大量表达,在分子量为13 kD处有一条与预测大小一致的特异性蛋白条带;经金属螯合柱纯化后,得到了电泳纯的融合蛋白.Western blotting检测结果表明:重组表达的融合蛋白与兔抗刀额新对虾MIH的多克隆抗体特异结合,证实该融合蛋白为中国明对虾MIH.通过胶内酶切与LC-ESI-MS分析进一步证实融合蛋白的部分肽段与日本对虾MIH一致.MIH融合蛋白的成功表达为进一步深入研究其在中国明对虾蜕皮过程分子作用机制奠定了基础.     

人免疫缺陷病毒I型p24gag基因的重组与表达

将编码人Ｉ型免疫缺陷病毒（ＨＩＶ－１）衣壳蛋白的ｐ２４ｇａｇ基因片段，克隆到原核表达载体ｐＥＴ－１７ｂ的Ｔ７噬菌体启动子下游，构建成了重组表达质粒ｐＥＴ２４，并使ｐ２４ｇａｇ基因片段在大肠杆菌ＢＬ２１（ＤＥ３）中高效表达，产物为３０ｋＤａ的ｓ１９－ｐ２４融合蛋白，表达量占菌体总蛋白的３８．４％。重组ｐ２４蛋白均与抗ｐ２４单克隆抗体及ＨＩＶ－１阳性血清发生特异性反应，具有较好的抗原性。     

鲤鱼(Cyprinus Carpio) 促性腺激素基因的克隆、原核表达及多克隆抗体制备

采用逆转录-聚合酶链式反应(RT-PCR)方法,从鲤鱼脑垂体获得了两种GtH β亚基的cDNA, 克隆在pMD18-T载体.经测序确证后,将这两个基因克隆到原核表达载体pET -32(a)中,转化E.coli表达菌BL21(DE3),以IPTG诱导融合蛋白的高效表达.利用初步纯化后的抗原免疫新西兰大白兔,制备多克隆抗体.应用制备的兔抗血清与抽提的鲤鱼脑垂体的总蛋白分别进行Western-blot及ELISA分析,结果显示获得的多抗能特异识别各自的天然蛋白.该结果为纯化天然GtH蛋白提供了有效的检测手段,为进一步制备GtH单克隆抗体奠定了基础.     

真鲷肿瘤坏死因子(TNFα)基因的体外重组与纯化研究

将真鲷肿瘤坏死因子(TNFα)基因的成熟肽区域克隆到表达载体pQE30中,并转化到大肠杆菌XL-blue中进行表达。转化子经载体和基因特异性引物进行PCR双向筛选,并将筛选到的阳性克隆经质粒纯化后,进行序列测定。序列测定结果显示,该基因结构正确,且准确插入到表达载体启动子的下游,获得了结构和组成正确的重组子。重、组子经IPTG诱导后,以SDS-PAGE电泳鉴定,电泳结果表明,该基因在大肠杆菌XL-blue中实现了表达。通过对诱导条件的调整,在体外获得了高效表达的重组蛋白,高效表达的重组蛋白约占菌体蛋白的25％-30％。不同诱导时间对重组蛋白影响实验显示,随诱导时间的增加,重组蛋白产量逐渐增高,经4h诱导,其表达量可以达到平台期,过长时间的诱导,对表达产量没有影响。重组蛋白经镍金属亲和层析纯化,获得了纯度较高的重组蛋白。采用交换缓冲液的方法,用Sephadex G150,对重组蛋白进行脱盐复性,使重组蛋白得到了进一步纯化。     

共表达H5、H7亚型AIV HA基因与鸡IL-18基因的重组鸡痘病毒构建

以鸡痘病毒(FPV)疫苗株为载体,将H5和H7亚型禽流感病毒血凝素(AIV HA)基因串联后(拥有同一个阅读框)和鸡白细胞介素-18(IL-18)基因分别插入到鸡痘病毒表达载体pUTA-16-LacZ复合启动子(ATI-P7.5×20)和单一启动子1(P7.5)下游,构建了携带AIV HA基因和鸡白细胞介素-18基因的重组鸡痘病毒转移载体质粒pUTAL-H5-H7-IL18;用相同的方法构建重组鸡痘病毒转移载体质粒pUTAL-H5-IL18;将H5亚型AIV HA基因插入到鸡痘病毒表达载体pUTA2复合启动子(ATI-P7.5×20)下游,构建了携带H5亚型AIV HA基因的重组鸡痘病毒转移载体质粒pUTA2-H5.应用脂质体转染法,将重组鸡痘病毒转移载体质粒与282E4株鸡痘病毒共转染鸡胚成纤维细胞(CEF),经BrdU进行三次加压蚀斑筛选后,以不同代次的细胞mRNA为模板,利用H5亚型AIV HA基因、H7亚型AIV HA基因和鸡IL-18基因特异引物进行RT-PCR和蛋白印迹检测,筛选出H5HA-H7HA融合蛋白基因和鸡IL-18基因共表达的重组鸡痘病毒rFPV-H5HA-H7HA-IL18,H5亚型AIV HA基因和鸡IL-18基因共表达的重组鸡痘病毒rFPV-H5HA-IL18以及单独表达H5亚型AIV HA基因的重组鸡痘病毒rFPV-H5HA.这些重组鸡痘病毒的构建为AIV活载体疫苗的研制奠定了基础.     

猪瘟病毒E2蛋白4重复抗原表位的构建及抗原活性研究

利用PCR方法获得4次重复的猪瘟病毒E2蛋白中和性抗原表位基因，将其克隆到pGEM-5ZF( )载体，测序正确后亚克隆到pGEX-3X载体构建得到重组质粒pGEX-3X-4P。重组质粒在大肠杆菌中诱导表达了含4重复抗原表位的融合蛋白。该蛋白经纯化后，利用间接ELISA检测其与血清的反应性，结果表明，纯化的融合蛋白与兔抗CSFV E2血清有很强的反应性，与兔抗BVDV E2血清不反应，说明该重复抗原表位在鉴别诊断CSFV与猪的BVDV感染方面具有潜在的应用价值。     

抗CD20嵌合抗体Fab'片段在大肠杆菌中高效表达

利用ＰＣＲ方法从抗ＣＤ２０ＳｃＦｖ表达载体上扩增重链可变区、轻链可变区基因,然后将ＶＨ、ＶＬ基因重组到Ｆａｂ’表达载体中,构建成抗ＣＤ２０嵌合抗体Ｆａｂ’片段表达载体ｐＹＺＦｃｄ２０,用ｐＹＺＦｃｄ２０转化大肠杆菌１６Ｃ９,在１６Ｃ９菌中分泌表达可溶性抗ＣＤ２０Ｆａｂ’片段,经分离纯化获得具有ＣＤ２０抗原特异结合活性的Ｆａｂ’片段,竞争性竞争荧光抑制实验表明,抗ＣＤ２０Ｆａｂ’片段竞争性抑亲本属源     

抗乙肝病毒表面抗原PreS1(20-47)的单链抗体在转基因烟草根分泌物中的初步表达

为探索利用植物根分泌表达重组蛋白的可行性，构建了含有抗乙肝病毒表面抗原PreS1(20—47)单链抗体(ScFv)基因的表达载体。该ScFv基因转化烟草后在烟草根部细胞的细胞质和内质网中获得表达。实验结果表明，5’端融合ER导向信号肽的重组ScFv可通过根分泌表达。     

密码子优化的牛精蛋白基因在大肠杆菌中的表达

以PCR的方法得到牛精蛋白基因的基因，去其内含子，得到牛精蛋白cDNA，克隆入原核表达载体pGEX-2T中，组装成表达载体pGEX-2T-PE，利用大肠杆菌偏好的编码精氨酸的密码子CGT将野生型牛精蛋白基因中编码精氨酸的稀有密码子（AGA或AGG）替换掉，通过基因合成得到密码子优化的牛精蛋白的基因，将其克隆入原核表达载体pGEX-2T中，组装成表达载体pGEX-2T-PS。将这两个表达载体分别转化入大肠杆菌表达菌株BL21中，经IPTG诱导，同样条件下，野生型牛精蛋白基因无法得到表达，密码子优化的牛精蛋白基因能够得到良好的表达，表达产物约占细菌总蛋白的18％，将表达蛋白纯化，进行DNA－蛋白结合实验，发现其能与DNA发生非特异性的结合。     

离子强度对于牛精蛋白在大肠杆菌中的表达的影响

通过基因合成将大肠杆菌偏好的精氨酸密码子CGT取代野生型牛精蛋白基因中的稀有精氨酸密码子AGA和AGG，得到密码子优化的牛精蛋白基因，并将其克隆入原核表达载体pGEX-2T中，组装成表达载体pGEX-2T-PS。将这个表达载体转化入大肠杆菌表达菌株BL21中，经IPTG诱导，可得到一33kD融合蛋白表达带，首次成功地将牛精蛋白在大肠杆菌中进行了表达，但其表达量很低，约为总菌体蛋白的13％。增加表遂菌株培养液的离子强度，可将蛋白表达含量提高到28％，且蛋白表达量与离子强度呈正相关。纯化后的牛精蛋白可在体外条件下与DNA发生结合。     

Determination of motility forces of bovine sperm cells using an "optical funnel"

An optical funnel, a new technique for the evaluation of the force of a microorganism, was applied to the determination of the motility force of bovine sperm cells. In this approach, sperm cells, suspended in an aqueous solution, are introduced into a flow cell, to which radiation pressure is applied from the direction opposite to a medium flow. The sperm cell, which is moving in a stream, is captured by radiation pressure and forced to move to the position at which the force induced by the laser radiation is equal to the force induced by a medium flow. The sperm cell then escapes by its own power on the way to this equilibrium (entrapping) position. The radiation force increases with decreasing distance from the focal point, and as a result, the force of the sperm cell can be determined by measuring the position where the sperm cell escaped against the laser irradiation field. The motility force of the sperm cell was measured in aqueous solution at different pH values and potassium ion concentrations. It was possible to measure more than 250 sperm cells in 3 h. Thus, the optical funnel has potential for use as a rapid and repetitive means for the determination of the motility force of the sperm cell.     

Modified PVA-Fe3O4 nanoparticles as protein carriers into sperm cells

Magnetite nanoparticles conjugated to protein are developed in order to potentially serve as protein carriers into bovine sperm cells. The conjugate comprises iron oxide nanoparticles that are covalently bound to an anti-protein kinase C (PKC)alpha antibody. This conjugate can serve for cellular PKC localization and the inhibition of its function. The surface of the nanoparticle is first modified with (3-aminopropyl) thrimethoxysilane to form a self-assembled monolayer, and subsequently conjugated with the antibody through amidation between the carboxylic acid end groups on the antibody and the amine groups on the surface of the nanoparticles. The anti-PKCalpha localization is proven by fluorescent microscopy and iron staining. The activity of the anti-PKCalpha conjugated with the nanoparticle is tested by recognizing PKCalpha using the Western blot method.     

Coarsening of needle-shaped apatite crystals in SiO2 • Al2O3 • Na2O • K2O • CaO • P2O5 • F glass

Coarsening of needle-shaped apatite crystals was studied in a SiO2 Al2O3 Na2O K2O CaO P2O5 F glass. Crystal numbers and size distributions were measured by image analysis of electron micrographs obtained from slightly etched fractures of powder compacts sintered from differently annealed glass powders. Results indicate that the growth of apatite needles is controlled by diffusion-limited Ostwald ripening. Thus, the mean needle diameter and length increase with t1/3 while the crystal number decreases with t–1 due to a constant volume fraction of apatite. An invariant reduced size distribution could be found for the diameters. The measurement of needle lengths is affected by several difficulties (e.g. random needle orientation).     

Performance Measurement in the Belgian Document Ordering and Delivery System Impala

This paper deals with performance measures and performance indicators in the Impala electronic document ordering and delivery system for research libraries in Belgium and compares these with some international standards as, e.g., the ProLib/PI study commissioned by the European Commission.Performance measures: Costs (clearinghouse principle) Number of ILL requests made to other libraries Number of ILL requests made to other libraries without success Number of ILL requests made to other libraries with success Number of ILL requests received from other libraries Number of ILL requests received from other libraries and not satisfied Number of ILL requests received from other libraries that were satisfied Frequency asked titlesPerformance indicators: Success rate Borrowing-lending ratio per library Response times, split into several segments of the ILL-procedureThe article concludes with some indications for quality measurement in electronic document delivery where Impala will be able to measure the real supply times as perceived by the end user.     

Spermicidal Activity of nonoxynol-9 and Analogs: Quantitative Assessments by Flow Cytometry

A flow cytometer-based method was developed for the quantitative assessment of spermicidal action of nonoxynol-9 (N-9) against human sperm. Two fluorescent dyes were chosen: carboxyfluorescein diacetate (CFDA) was employed as an indicator for viable sperm, while propidium iodide (PI) identified the sperm membrane integrity disrupted by spermicidal agents. Living, motile sperm were identified by the green fluorescence of CFDA, while the red fluorescence of PI reflected the ability of the spermicide to kill sperm. Both living and dead sperm were effectively resolved from the acellular components of seminal fluid. N-9 was used as a model spermicidal agent to study the feasibility as well as establish the various technical aspects of this method. Further studies with a series of structurally-related nonoxynol analogs, using the method established, demonstrated that N-9 is the most effective spermicidal agent. The spermicidal activity of N-9 and analogs was also determined by a a computer-assisted semen analysis (CASA) method for comparison. This conventional CASA method is designed to detect sperm motility, in contrast to the flow cytometry method which is capable of identifying the loss of membrane integrity. A discrepancy in the concentration needed for comparable spermicidal action between these assay methods was observed, which could imply that there is a concentration dependency on the disruption of membrane integrity and the complete loss of motility.     

Special dilution refrigerator systems for milli-Kelvin detector experiments

Several hundred ultra low temperature systems have been designed and built for a variety of applications. One common application is the refrigeration of low temperature detectors. Although many of the requirements are satisfied by our standard designs, we have often built special refrigerators to suit specific detector requirements. A few of the most interesting of these systems will be discussed. dilution refrigerators to cool gravitational wave antennae to 65 mK rotating dilution refrigerator for cosmic ray detector experiments compact dilution refrigerator to cool large bolometer arrays within the SCUBA telescope side access systems for beam line experiments