甘露聚糖结合凝集素相关丝氨酸蛋白酶

甘露聚糖结合凝集素（MBL）是一种重要的天然免疫防御分子,通过激活MBL相关丝氨酸蛋白酶（MAST）而启动补体激活凝集素途径来清除病原体。本文就MASP的基因、分子结构及功能等方面研究概况作一介绍。     

大鼠丝氨酸或半胱氨酸蛋白酶抑制剂B2的表达、纯化及抗体制备

目的:原核表达、纯化大鼠丝氨酸或半胱氨酸蛋白酶抑制剂B2(SERPINB2),并制备其多克隆抗体.方法:设计扩增大鼠Serpinb2全长基因的特异引物,通过PCR扩增出该基因片段,测序正确后插入含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克...     

棉铃虫组织蛋白酶B多克隆抗体制备及鉴定

介绍了用重组的棉铃虫组织蛋白酶B(HCB)制备兔多克隆抗体的方法。用已经克隆得到的pGEX 4T 1 HCB重组质粒转化的大肠杆菌 (Escherichiacoli)BL2 1菌株进行诱导表达 ,获得融合表达的包涵体产物。经过变性、复性等方法处理包涵体 ,获得可溶性融合蛋白。用凝血酶裂解融合蛋白 ,再经SDS -聚丙烯酰胺凝胶电泳分离纯化目的蛋白HCB ,用做抗原免疫家兔。产生的抗血清经硫酸铵沉淀 ,最后得到了抗棉铃虫组织蛋白酶B的多克隆抗体。用间接酶联免疫吸附测定法测得该抗体对重组表达的组织蛋白酶B和棉铃虫体内的组织蛋白酶B的效价分别为 1∶5 1 2 0 0和 1∶2 5 60 0。通过免疫印迹检测证明此抗体不仅可以识别重组表达的棉铃虫组织蛋白B ,而且可以识别棉铃虫卵巢匀浆液中的棉铃虫组织蛋白酶B。     

家蚕丝氨酸蛋白酶抑制剂4(serpin-4)的基因克隆、原核表达和多克隆抗体制备

丝氨酸蛋白酶抑制剂4(serpin-4)为丝氨酸蛋白酶抑制剂家族中的一员.本研究旨在研制高效价的家蚕Bombyx mori serpin-4多克隆抗体,为深入研究serpin-4基因的生理功能打下物质基础.首先于家蚕脂肪体中克隆了serpin-4基因,利用基因重组技术构建了pET28a-serpin-4原核表达载体,经...     

抗欧亚活血丹凝集素特异性多克隆抗体的制备

欧亚活血丹外源凝集素(Gleheda)是分离自欧亚活血丹 (Glechoma hederacea) 叶片中的一种糖基化植物新蛋白. 如同其他糖基化蛋白,通过免疫学方法探测 Gleheda 的过程中通常受到一些不相干糖蛋白的妨碍,为此制定了抗 Gleheda 特异性多克隆抗体的纯化方案. 免疫血清蛋白经硫酸铵选择性沉淀后,分别以 Gleheda 和刺槐外源凝集蛋白 (RPA) 结合在 Sepharose 4B作为亲和配体,采用亲和层析法连续纯化 2 次,然后进一步采用离子交换层析 Q Fast Flow 提纯. 经每一步骤提纯得到的抗体组分对 Gleheda 的特异性,均同时采用双向免疫扩散检验和 Western blot 分析. 结果表明,以 Gleheda 为配体,亲和纯化制备得到的抗体组分对叶片粗提物中的许多植物 (糖) 蛋白仍然表现交叉反应. 为除去由植物糖蛋白中的聚糖所引起这些非特异性交叉反应抗体,接着以 RPA 为配体再次进行亲和纯化,Western blot 分析显示,抗体的特异性得到提高但并非除去了所有非特异性交叉反应的抗体. 最后进一步采用离子交换层析制备得到仅抗 Gleheda 蛋白的特异性抗体组分,此抗体组分适用于免疫探测研究. 该抗体纯化制备程序简易而高效,而且不需要昂贵的设备.     

非洲爪蟾PAPC多克隆抗体的制备及其特异性鉴定

非洲爪蟾ParaxialProtocadherin(PAPC)是一个在爪蟾Spemann组织者特异表达的膜蛋白.它在爪蟾原肠运动阶段的汇聚延伸运动和体节发生阶段的体节边界形成,以及早期听泡的形态发生和细胞特化过程中都有重要的作用.为了研究PAPC基因在早期胚胎发育过程中的表达及其生物学功能,需要制备PAPC抗体.应用谷胱甘肽S-转移酶(glutathioneStransferase,GST)表达系统表达GST-PAPC融合蛋白,亲和纯化后用以免疫新西兰大白兔,获得PAPC多克隆抗体.免疫印迹分析发现,以1∶3000稀释的该多克隆抗体为一抗时,能够在转染了全长PAPC质粒的HEK293T细胞的蛋白质抽提物中,特异地识别出150ku的印迹条带.同时,GST-PAPC融合蛋白可以竞争性抑制该抗体对全长PAPC质粒转染细胞的蛋白质抽提物的特异性条带.用1∶500稀释的该抗体为一抗进行免疫荧光分析时,发现,PAPC多克隆抗体能够识别在HEK293T细胞中过表达以及爪蟾动物极细胞中过表达的PAPC蛋白,荧光信号定位在细胞膜上.免疫印迹分析证明,PAPC抗体能够识别爪蟾胚胎中内源表达的PAPC蛋白.     

小鼠着床丝氨酸蛋白酶2cDNA的克隆、表达及抗体制备

以孕 4 .5天小鼠子宫着床位点总cDNA为模板 ,用PCR方法扩增获得着床丝氨酸蛋白酶 2 (ISP2 )的cDNA。序列分析表明 ,该cDNA序列中的开放阅读框编码 2 79个氨基酸 ,并与文献报道完全相同 ,但在 3′非翻译区多 2 0 4bp ,已登录于GenBank(登录号 :AF4 4 2 819)。为了获得重组ISP2 (rISP2 ) ,构建了pGEX 4T 2 ISP2表达质粒 ,进而在大肠杆菌BL2 1(DE3)中表达融合蛋白质GST ISP2 ,经SDS PAGE回收的融合蛋白质用牛凝血酶酶切 ,再经SDS PAGE回收获得rISP2。用rISP2作为免疫原免疫家兔获得ISP2多克隆抗血清。用该抗血清进行免疫组化研究表明 ,ISP2在妊娠早期的小鼠子宫内膜腺上皮有特异性的高表达。     

PBP2a多克隆抗体制备及其乳胶凝集检测法的建立

目的制备兔抗青霉素结合蛋白2a(penicillin binding protein 2a,PBP2a)抗体,建立检测PBP2a的乳胶凝集法。方法以重组PBP2a转肽酶区蛋白免疫家兔制备多克隆抗体,ELISA和Western blot法检测所制备的抗血清效价和特异性,用纯化的多抗建立乳胶凝集法。结果纯化的重组蛋白免疫家兔能有效地刺激特异性抗体的产生,抗血清的效价达1∶25 600,Western blot显示该抗体能有效识别原核表达及MRSA临床分离株中的PBP2a,建立了乳胶凝集法,敏感性及特异性良好。结论成功制备了抗PBP2a抗体血清,初步建立了检测PBP2a的乳胶凝集法,为有效制备高特异的单克隆抗体进而研制MRSA快速鉴定试剂盒奠定了良好的基础。     

D1蛋白酶的克隆表达、纯化、生物活性测定及多克隆抗体的制备

为了开发以D1蛋白酶作为靶标的新型除草剂,需要对先导化合物的生物活性进行检测和筛选。从菠菜中提取总RNA,逆转录合成cDNA,采用PCR扩增了CtpA的基因,连接至表达载体pET-28a中,构建了重组表达质粒,并在大肠杆菌BL21(DE3)中进行高效表达,通过降低IPTG和诱导温度获得了可溶性表达的重组蛋白酶。采用Ni-NTA亲和层析和凝胶过滤柱层析对重组蛋白进行了纯化,SDS-PAGE和Western blotting结果证实目的蛋白为含有His-tag的融合蛋白。以合成的D1前体蛋白羧基端24肽作为模拟底物,采用高效液相色谱法测定了其水解活性,结果表明活性可达1.10nmol/(mg·min),为文献报道数据的15倍。纯化后的CtpA蛋白免疫日本的长耳大白兔制备多克隆抗体,ELISA法测定其血清抗体的效价高达1:100000。该结果为抑制剂先导化合物的筛选和酶蛋白与化合物的作用机理研究提供了必要的基础。     

梅花鹿重组Galectin-1的表达及多克隆抗体的制备与鉴定

半乳糖凝集素-1（Galectin-1）是最先被报道的哺乳动物半乳糖凝集素,存在于多种组织和细胞内,参与细胞的粘附、增殖、凋亡和炎症反应等多种生理病理过程,并且与免疫系统的调节和肿瘤的发生发展密切相关。鹿茸是哺乳动物中罕见的能够周期性的脱落和再生的附属器官,作为研究哺乳动物器官再生的新模型受到关注。鹿茸再生是一个基于干细胞的过程,定位于角柄骨膜的干细胞是鹿茸再生的基础,Galectin-1在角柄骨膜细胞（pedicle periosteum cell, PPC）中高度表达,提示其在鹿茸再生中发挥着重要的作用。由于尚没有商用的鹿Galectin-1蛋白及其抗体,为进一步研究Galectin-1在鹿茸再生中的生物学功能,需要制备相应的蛋白和抗体,本实验将梅花鹿Galectin-1基因与pET28a连接,并将重组质粒pET28a-Galectin-1转入大肠杆菌（Escherichia coli）BL21(DE3)中诱导表达。用Ni-NTA Agarose亲和层析纯化融合蛋白,免疫兔子制备多克隆抗体。酶联免疫吸附（enzyme-linked immunosorbent assay, ELISA）法检测抗体效价、Western blot 检测抗体特异性、细胞免疫荧光检测Galectin-1在角柄骨膜细胞中的表达情况。结果表明,本实验成功诱导重组原核表达载体pET28a-Galectin-1在BL21(DE3)中表达,通过Ni纯化获得融合蛋白。ELISA结果显示,抗体效价达到1:64000,Western blot结果表明该抗体特异性良好,细胞免疫荧光显示Galectin-1在PPC全细胞中表达。本实验获得了纯化的鹿Galectin-1蛋白和特异性较好的多克隆抗体,为揭示Galectin-1在鹿茸再生调控中的作用提供了重要的实验材料。     

卡他莫拉菌UspA1蛋白多克隆抗体的制备及鉴定

为制备抗卡他莫拉菌(Moraxella catarrhalis,Mc)表面蛋白UspA1胞外结构域的多克隆抗体(PcAb),对UspA1蛋白进行生物信息学分析,获取胞外结构域中抗原表位最为丰富的肽段,找到其对应的基因序列并引入大肠杆菌偏好性密码子,对其优化后化学合成全基因序列。将该基因序列按常规方法克隆入表达载体p ET-28a(+)后表达重组UspA1-His融合蛋白并纯化。以该纯化抗原免疫新西兰大白兔,经4次免疫后,用Protein A亲和层析柱从抗血清中纯化出抗UspA1-His融合蛋白PcAbIgG。经免疫荧光法、酶联免疫吸附法及Western blotting鉴定,抗UspA1-His融合蛋白PcAb能特异性识别UspA1蛋白的表面暴露区。该多抗的制备为下一步建立卡他莫拉菌快速检测技术奠定了基础。     

抗血管紧张素原多克隆抗血清的制备及鉴定

为深入研究血管紧张素原（angiotensinogen,AGT)的表达和组织分布特征,我们以原核表达的AGT蛋白C末端片段为免疫原免疫家兔,制备了针对AGT的多克隆抗血清,并对其进行了ELISA、Western印迹分析及免疫组化鉴定。结果发现,经多次免疫后获得的多克隆抗血清不仅效价高（1：25600）,而且能特异性地针对组织内源性及原核表达的AGT蛋白,同时在人和大鼠组织AGT之间会发生交叉反应。以此抗血清进行免疫组化染色,观察到人胰腺的胰岛细胞及杆内胆管上皮细胞胞浆均有AGT蛋白表达。以上结果提示,利用大鼠杆菌融合表达的AGT蛋白可有效刺激家兔产生AGT抗体。本工作为进一步研究AGT乃至局部RAS的生理、病理意义提供了重要的实验工具。     

Preparation and characterization of polyclonal antibodies against human chaperonin 10

Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.     

HIV-1辅助蛋白Vpr多肽抗体的制备与鉴定

预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。     

Preparation and characterization of polyclonal and monoclonal antibodies against the insecticide DDT

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared anti-DDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/1 were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site.     

抗博尔纳病病毒核蛋白多克隆抗体的制备和鉴定

目的利用重组博尔纳病病毒核蛋白进行动物免疫,制备多克隆抗体并对其进行鉴定。方法将重组载体pET14b-p40转化至感受态大肠埃希菌I,PTG诱导融合蛋白的表达,His-tag亲和层析纯化重组核蛋白并作为抗原免疫新西兰大白兔,收集免疫后血清,制备和纯化多克隆抗体,ELISA测定抗体效价,并进行Western-blot鉴定。结果成功制备出核蛋白多克隆抗体,ELISA检测效价高达1︰256000;该抗体与原核和真核系统中表达的核蛋白均能发生特异性反应。结论成功制备了效价和特异性良好的抗重组核蛋白多克隆抗体,为博尔纳病病毒血清免疫学检测方法的建立奠定了基础。     

Cross-reactivities of polyclonal antibodies against factor V activating enzyme, a serine proteinase from Vipera lebetina (snake) venom

Antibodies to the factor V activating enzyme from Vipera lebetina venom were produced by immunizing a rabbit with chromatographically purified factor V activating enzyme probes. The antibodies cross-reacted with different protein fractions in 23 snake venoms (ten viperid, eight crotalid, and five elapid venoms) as demonstrated by western immunoblotting. In the venom of Vipera russelli the antibodies recognized only one protein band which probably belonged to factor V activating enzyme.     

棉铃虫Hsp70的多克隆抗体制备及鉴定

HSPs(热休克蛋白)是机体在不利环境条件刺激下合成的一类蛋白质,在进化上高度保守,普遍存在于生物体,其中Hsp70是最为保守的成员。研究发现,昆虫滞育过程中普遍存在Hsp70表达上调的现象。然而,现有的研究均是在基因水平的检测结果。为了能从蛋白水平检测棉铃虫中Hsp70的表达,本研究制备了棉铃虫Hsp70的多克隆抗体。构建了hsp70的原核表达载体并在大肠杆菌BL21(DE3)中成功表达,重组蛋白经镍柱纯化后免疫兔子,制备了棉铃虫Hsp70的多克隆抗体。抗体的效价较高,达到了1∶256 000,此外Western结果表明,制备的抗体能检测出热诱导的Hsp70蛋白。本研究制备了高效价且较为特异的Hsp70对克隆抗体,该抗体为后续从蛋白水平研究棉铃虫滞育过程中Hsp70的表达及其分子机制奠定了基础。     

Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens

A method is described for obtaining from plants partially purified preparations of mycoplasma-like organisms (MLO) which are suitable for use as immunogens for polyclonal or monoclonal antibody production, and as antigens for directly coating ELISA plates. Using this method a mouse monoclonal antibody to primula yellows MLO was prepared, and its characteristics compared with those of primula yellows polyclonal antibodies from rabbits and also against polyclonal antibodies made to similar preparations of European aster yellows MLO. No serological distinction was obtained between any of the homologous or heterologous combinations of antibody and MLO preparation using ELISA, fluorescence microscopy with FITC-labelled antibodies, or immunoprobes of western blots of partially purified MLO preparations. By contrast, there were no cross-reactions between the primula or aster yellows antibodies or MLO preparations and preparations of clover phyllody or tomato big bud MLOs or their respective polyclonal antibodies. The primula yellows MLO monoclonal and polyclonal antibodies, and also the European aster yellows MLO polyclonal antibodies, all appeared to recognize only a single major antigen of approximate M, = 22 400 daltons. Some possible explanations for the apparent specificity of the polyclinic antisera for a single antigen, and the relevance to MLO preparation procedures are discussed.     

SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)