利用噬菌体表面展示技术筛选转铁蛋白黏附肽的研究

为了筛选转铁蛋白黏附肽,应用噬菌体表面展示技术经过三轮生物淘选,成功地从随机七肽库中得到黏附转铁蛋白的重组噬菌体克隆,经过相对亲和力常数测定和DNA测序得到4个转铁蛋白黏附肽的序列。实验中以回收率和选择比为操作参数,对淘选进行了优化,并发展了一种基于噬菌体滴度的相对亲和力常数测定方法。转铁蛋白受体是一种有效的肿瘤标记物,利用转铁蛋白为载体可以实现药物靶向运输,因此转铁蛋白黏附肽将是重组蛋白质药物连接转铁蛋白的有用标签。     

Rapid Preparation of Stable Isotope Labeled Peptides that Bind to Target Proteins by a Phage Library System

We have developed a system for directly isolating foreign peptides displayed on the N-terminus of the major coat protein of bacteriophage M13. The phage particle in this system is formed as a mixture of wild type and modified coat proteins. The N-terminal segment of the modified coat protein was mutated for chemical cleavage, in order to obtain the displayed peptide from the major coat protein. Using ¹³C, ¹⁵N- labeled medium, we introduced stable isotopes, ¹³C and/or ¹⁵N, into the coat proteins. The NMR spectra for the cleaved peptides from the phage particles could be recorded within a few days after the selection of the phage clone.     

脑膜炎大肠杆菌IbeA蛋白结合肽序列的亲和筛选

应用噬菌体展示肽库技术,以重组的脑膜炎大肠杆菌致病蛋白IbeA作为靶分子,经过吸附-洗脱-扩增-再吸附的亲和筛选,随机挑选亲和力强的噬菌体克隆,进行ELISA、竞争抑制实验和序列测定。结果显示,经3轮淘选后,间接ELISA鉴定得到高亲和性结合IbeA蛋白的15个阳性克隆。竞争抑制实验结果表明,游离IbeA蛋白能竞争抑制噬菌体结合肽克隆与固相包被的IbeA蛋白的结合,其抑制作用随游离IbeA蛋白浓度的降低而减弱。测序结果得到5种阳性噬菌体克隆展示肽序列。上述结果提示以脑膜炎大肠杆菌IbeA蛋白为靶筛选所获得的噬菌体12肽克隆,具有特异性,其结合肽序列呈现相对保守性。建立的从噬菌体随机肽库筛选IbeA蛋白结合肽的方法具有方便、灵活和高效可行的特点。     

利用噬菌体肽库筛选与HIV-1p24抗原结合的多肽

通过噬菌体展示技术筛选出与HIV-1p24抗原结合的多肽,为用多肽辅助p24抗原检测提供实验基础.以重组p24抗原为靶蛋白,对噬菌体随机七肽库进行三轮筛选,用EUSA鉴定第三轮筛选到的噬菌体克隆与p24重组抗原的结合能力,再对噬菌体克隆进行测序分析,同时研究了ELISA中噬菌体加入量及多种封闭剂对噬菌体特异性结合能力的...     

应用噬菌体肽库筛选能封阻霍乱噬菌体VP1转染菌细胞的寡肽

噬菌体感染细菌首先要吸附于细菌表面受体 ,从目前报道的细菌与噬菌体相互作用的研究中发现 ,这些受体包括细菌细胞外膜上的蛋白、糖脂结构和鞭毛等。霍乱弧菌是霍乱的病原体 ,高守一等 (副霍乱资料汇编 ,1 984,2 37～ 2 4 5 .)从国内分离并选择出 5株噬菌体 (VP1～VP5 ) ,根据霍乱弧菌菌株对噬菌体的敏感性不同 ,将埃尔托型霍乱弧菌分为 32个噬菌体型。结合生物学分型方法 ,可区分埃尔托型霍乱弧菌的两类不同菌株 (流行株和非流行株 )和不同菌型。对各种来源的菌株进行分型 ,可作为一种追溯传染来源、传播途径和分析流行形式的流行病学研…     

Botulinum Neurotoxin Devoid of Receptor Binding Domain Translocates Active Protease

Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ∼50 kDa light chain (LC) protease is translocated into the cytosol by the ∼100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication.     

Screening of Peptides that Inhibit Bacterial Binding to Fibronectin using Combinatorial Peptide Libraries

Infective endocarditis is often caused by passage of oral endogenous bacteria into the blood stream. Such bacteremia occurs after tooth extraction, and occasionally even after brushing of the teeth. Abnormal or damaged heart valves, including artificial valves, show high risk of infection. Antibiotics are widely used to prevent this infection, however, frequent use of these have resulted in the generation of resistant mutants, which generate serious social problems. Thus, the development of more effective and safer drugs for the prevention of such infections is very desirable. The adhesion of bacteria to fibronectin, one of the major extracellular matrix (ECM) protein exposed on the wound endocardia, is considered critical for the infection. We have previously found a novel mode of interaction between endocarditis-causing bacteria and human fibronectin. The present study focuses on the discovery of candidate compounds that inhibit the association between microorganisms and fibronectin. Positional scanning libraries (PSL) with N-terminal biotinylated 6-mer peptides have been constructed and screened for binding to a monoclonal antibody for fibronectin that inhibits the bacterial fibronectin-binding. The consensus sequences derived from these experiments are expected to be structural mimetics of the local structure of fibronectin involved in the bacterial adhesion. Since individual synthetic 6-mer peptides did not show the desired action, discontinuous epitopes can be envisaged and therefore a 9-mer-PSL was constructed to reveal conformational epitopes. In the second library, several 9-mer peptides based on the screening were synthesized and gave improved results.Australian Peptide Conference Issue     

泰泽氏病原体抗原表位相关肽的初步筛选与鉴定

目的获得泰泽氏病原体抗原表位相关肽,用于实验动物血清中该病原体感染相关抗体的检测。方法选用泰泽氏病原体的四种单克隆抗体(M2、M3、M4、M5)作为配基,从噬菌体表面展示的随机7肽文库中筛选单抗识别的抗原表位,获得特异性噬菌体克隆;并采用ELISA、Western blot方法对其进行分析鉴定,获得阳性噬菌体克隆。结果获得阳性噬菌体克隆5个,其展示的融合蛋白能被泰泽氏病原体的免疫血清识别,ELISA检测A值的P/N为8.0～17.1;Western blot分析显示单一特异性条带,相对分子质量约为38×103。结论本研究获得的5个阳性克隆所表达的融合蛋白,为泰泽氏病原体抗原表位相关肽,可作为该病原体隐性感染血清学检测的候选抗原。     

Screening of a Small Set of Random Peptides: A New Strategy to Identify Synthetic Peptides that Mimic Epitopes

Small diversity libraries, composed of 4550 synthetic dodecapeptides and 8000 synthetic tripeptides, have been used to identify sequences homologous to small linear and non-linear parts of epitopes. Here we report that synthetic peptides identified through alignment of dodecapeptides and tripeptides derived from these small libraries have, in direct ELISA and/or competitive ELISA, activities similar to that of peptides covering the native epitope and similar to that of peptides derived from large expression libraries composed of 10⁶–10⁷ random peptides. This result was obtained with the monoclonal antibodies 6A.A6 and M2. Mab 6A.A6 binds the transmissible gastroenteritis virus (TGEV) and mAb M2 binds the FLAG^®-peptide, an affinity tag. It was also found that the antibody binding activity of peptides, derived from small or large libraries, can strongly depend on the way in which the peptide is presented to the antibody, i.e. high antibody titers were obtained when these peptides were synthesized on pins or coated onto microtiter plates, whereas low IC₅₀s were obtained with these peptides in solution. We postulate that small peptide libraries may be a powerful tool to quickly identify new peptides that can be used as sensitive markers for mAbs of interest. © 1997 John Wiley & Sons, Ltd.     

Screening and Identification of the Binding Peptides of Mycoplasma genitalium Protein of Adhesion

International Journal of Peptide Research and Therapeutics - Mycoplasma genitalium protein of adhesion (MgPa) is a vital membrane protein, which plays an important role in mediating the adhesion...     

Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-Å X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.     

Construction of an Artificially Randomized IgNAR Phage Display Library: Screening of Variable Regions that Bind to Hen Egg White Lysozyme

To develop a multi-antigen-specific immunoglobulin new antigen receptor (IgNAR) variable (V) region phage display library, CDR3 in the V region of IgNAR from banded houndshark (Triakis scyllium) was artificially randomized, and clones specific for hen egg white lysozyme (HEL) were obtained by the biopanning method. The nucleotide sequence of CDR3 in the V region was randomly rearranged by PCR. Randomized CDR3-containing segments of the V region were ligated into T7 phage vector to construct a phage display library and resulted in a phage titer of 3.7?×?10⁷ PFU/ml. Forty clones that contained randomized CDR3 inserts were sequenced and shown to have different nucleotide sequences. The HEL-specific clones were screened by biopanning using HEL-coated ELISA plates. After six rounds of screening, nine clones were identified as HEL-specific, eight of which showed a strong affinity to HEL in ELISA compared to a negative control (i.e., empty phage clone). The deduced amino acid sequences of CDR3 from the HEL-specific phage clones fell into four types (I?IV): type I contains a single cysteine residue and type II?IV contain two cysteine residues. These results indicated that the artificially randomized IgNAR library is useful for the rapid isolation of antigen-specific IgNAR V region without immunization of target antigen and showed that it is possible to isolate an antigen-specific IgNAR V region from this library.     

以表位模拟肽为亲和靶标的肉毒毒素受体结合抑制剂的筛选及鉴定

以设计合成的A型肉毒毒素表位模拟肽为亲和靶标,对噬菌体随机肽库进行筛选,寻找能与A型肉毒毒素表位模拟肽特异结合并能拮抗毒素毒性效应的分子,通过ELISA鉴定阳性克隆,并对鉴定的阳性克隆进行特异性分析及DNA测序。氨基酸序列同源性分析发现,针对P4、P5表位模拟肽获得了两条特异的结合序列,并通过动物保护实验在噬菌体展示肽水平对特异结合分子的毒素毒性拮抗效应进行了初步研究,初步证明结合肽对A型肉毒毒素有一定的保护作用。     

脓毒症单核巨噬细胞特异性结合肽的筛选

利用噬菌体随机肽库展示技术,筛选出与脓毒症单核/巨噬细胞特异性结合的短肽,探索脓毒症治疗的新方法.分别以经过脂多糖(lipopolysaccharide, LPS)处理的人外周血单核细胞株(THP-1)细胞作为筛选的靶细胞,以未经LPS处理的THP-1细胞作为非特异性噬菌体吸附细胞,对噬菌体随机环七肽库进行4轮“差减"筛选,经过细胞ELISA验证阳性噬菌体克隆,对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体克隆与LPS处理THP-1细胞的结合特异性.4轮筛选后,随机挑取的噬菌体克隆,测序后得到可与LPS处理的THP-1细胞特异性结合肽.对去冗余后的七肽进行Clustal W多序列比对分析和BlastP蛋白同源相似性分析,细胞免疫荧光检测确定获得的噬菌体展示七肽可与LPS处理的THP-1细胞特异性结合.噬菌体随机肽库技术为脓毒症单核/巨噬细胞表面靶位的筛选提供了高效、快捷的筛选体系,实验获得的多肽基序具有高度保守性和细胞特异性,这些多肽的生物活性将是下一步的研究内容.     

Purification and Characterization of Nontoxic Protein Complex from Serotype D 4947 Botulinum Toxin Complex

The large-sized botulinum toxin complex (L-TC) is composed of botulinum neurotoxin (BoNT) and nontoxic proteins, e.g. nontoxic nonhemagglutinin (NTNHA) and three types of hemagglutinins (HAs; HA-33, HA-17 and HA-70). The nontoxic proteins play a critical role in L-TC oral toxicity by protecting the BoNT in the digestive tract, and facilitating absorption of the L-TC across the intestinal wall. Under alkaline conditions, the L-TC separates into BoNT and the nontoxic protein complex (NC). In this study, we established a two-step procedure to yield highly pure NC from the L-TC produced by Clostridium botulinum serotype D strain 4947 in which the NC was isolated from the L-TC by gel filtration under alkaline conditions followed by immunoprecipitation with an anti-BoNT antibody to remove contaminating BoNT from the NC fraction. Western blotting and electrophoretic analysis showed that the highly purified NC fraction had only very slight or no BoNT contamination. In addition, the purified NC fraction showed no intraperitoneal (ip) toxicity to mice at a dose of 38?ng per animal whereas the L-TC exhibited an ip median lethal dose of 0.38?ng per mouse. The highly purified NC displayed the same hemagglutination titer as the L-TC. The NC, as well as the L-TC, demonstrated cell binding and monolayer transport in the rat intestinal epithelial cell line IEC-6. Consequently, the highly purified NC can function as a ??delivery vehicle?? even without the BoNT.     

Analytical Ultracentrifugation Studies of Phage ?29 Protein p6 Binding to DNA

Protein p6 from Bacillus subtilis phage ?29 binds double-stranded DNA, forming a large nucleoprotein complex all along the viral genome, and has been proposed to be an architectural protein with a global role in genome organization. Here, we have characterized quantitatively the DNA binding properties of protein p6 by means of sedimentation velocity and sedimentation equilibrium experiments permitting determination of the strength and stoichiometry of complex formation. The composition dependence of protein binding to DNA is quantitatively consistent with a model in which the protein undergoes a reversible monomer-dimer self-association, and the dimeric species binds noncooperatively to the DNA. We also have found that when the anisotropic bendability periodicity of the nucleotide sequence preferred by p6 is modified, nucleocomplex formation is impaired. In addition, suppression of complex formation at high ionic strength is reversed by the addition of high concentrations of an inert polymer, mimicking the crowded intracellular environment. The results obtained in this work illustrate how macromolecular crowding could act as a metabolic buffer that can significantly extend the range of intracellular conditions under which a specific reaction may occur.     

人类天然抗α-Gal抗体抑制肽的筛选

人类天然抗体可以与猪细胞抗原表位Gal-α1,3-Gal结合,触发超急性排斥反应（HAR）,HAR是猪器官移植至人体时的首要障碍.阻断人的天然抗体,阻止其与猪细胞表面特异性抗原的结合,是防止超急性排斥的有效措施.利用噬菌体展示技术,从XCX₁₅随机肽库中筛选与西非单叶豆凝集素(GS-I-B₄）特异性结合的噬菌体展示肽.得到一个小肽序列为SCTALSFPSFAFLARGT,其与人血清中天然抗体的结合可以被蜜二糖（melibiose）竞争性地抑制,同时该小肽还能抑制人类天然抗体介导的猪红细胞的凝集反应.因此,筛选到的小肽能作为人天然抗α-Gal抗体的抑制肽.     

Cloning,Expression, Purification,and Characterization of Biologically Active Recombinant Hemagglutinin-33, Type A Botulinum Neurotoxin Associated Protein

Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.     

一种用于穿透多肽筛选的随机文库的构建及筛选

以增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)为示踪物,在pET-14b载体上构建编码12个氨基酸的随机多肽表达文库.建立一种简便、经济、有效的文库筛选方法,从所构建的文库中筛选出细胞穿透多肽（cell-penetrating peptide, CPP）. 采用点突变技术,首先在pET-14b载体多克隆位点NdeⅠ和XhoⅠ之间加入4个限制性内切酶位点,随后在BamH Ⅰ位点后加入三联终止密码子,接着再利用亚克隆的方法在Kpn Ⅰ 和XhoⅠ之间插入EGFP,形成一个新的用于原核表达示踪蛋白的载体pET-14bMCStop/EGFP.最后再利用点突变技术在上述构建的示踪载体的多克隆位点XhoⅠ和BamH Ⅰ之间插入36个随机碱基序列.以His-Tat-EGFP作为工具建立有效的筛选方法,利用这种方法对文库进行筛选. 酶切和测序表明,示踪载体的构建是正确的,且在大肠杆菌中可有效地表达出His标记的EGFP.在示踪载体的基础上构建的随机多肽文库至少包含了10⁵个独立克隆,其中90%以上的克隆插入的随机片段都是36个碱基.建立的筛选方法是可行的,并用此方法进行了初步的筛选.