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.     

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。     

人源抗ICAM-1单链抗体的制备及其生物学活性鉴定

利用噬菌体展示技术筛选特异性人源抗ICAM-1单链抗体（Anti-human ICAM-1 scFv）并进行生物学活性鉴定。应用Tomlinson I+J噬菌体抗体库,以P1抗原肽为包被抗原,经过4轮“吸附-洗脱-扩增”进行亲和富集筛选。以PCR反应、ELISA抗原交叉反应和Dot blotting实验进行阳性克隆的鉴定。scFv经原核表达和分离纯化后,以Western blotting实验、竞争ELISA实验和细胞黏附抑制实验对其生物学活性进行初步鉴定。Tomlinson I+J噬菌体抗体库经4轮亲和富集筛选,利用ELISA方法成功筛出4株阳性克隆。通过PCR鉴定反应、ELISA抗原交叉反应和Dot blotting实验,最终获得了1株既能与P1抗原肽特异结合又能与人ICAM-1抗原特异结合的阳性克隆J-A1。对scFv进行原核表达和亲和层析后获得了高纯度的目的蛋白。竞争ELISA实验和细胞黏附抑制实验证实纯化的scFv具有良好的亲和活性和抗细胞黏附活性。文中成功利用噬菌体展示技术筛选到特异性人源抗ICAM-1 scFv,为进一步探索该抗体在炎症相关性疾病治疗中的应用奠定了基础。     

噬菌体肽库筛选抗人B7-H4中和抗体的模拟抗原表位及其免疫原性鉴定

目的:用噬菌体呈现随机12肽库筛选能与抗人B7-H4(h B7-H4)中和抗体特异性结合的模拟抗原表位肽,并用其免疫小鼠检测其免疫原性。方法:以抗h B7-H4中和抗体为靶分子,用体外生物淘洗法从噬菌体呈现随机12肽库中筛选与之结合的噬菌体克隆,用竞争性细胞ELISA鉴定阳性噬菌体克隆;化学合成候选多肽,并与钥孔血蓝蛋白或破伤风毒素偶联鉴定多肽的特异性;进一步用融合蛋白免疫小鼠检测其免疫原性和抗血清的补体依赖的细胞杀伤活性(CDC)。结果:经过3轮体外筛选后随机挑取50个阳性噬菌体克隆,其中20个克隆与抗h B7-H4抗体有较强的结合能力,DNA测序得到6组结构相似的肽序列;竞争性ELISA结果显示1号肽噬菌体能与细胞表面的h B7-H4竞争性地结合抗h B7-H4单抗;点杂交结果显示1号肽能特异性结合抗h B7-H4单抗;小鼠免疫实验结果显示1号肽融合蛋白能诱导高滴度的抗h B7-H4抗血清,并且抗血清具有补体依赖的细胞杀伤活性。结论:筛选得到能与抗h B7-H4中和抗体特异性结合的12肽模拟抗原表位序列并且具有免疫原性,为进一步开发h B7-H4相关的多肽疫苗提供了实验依据。     

表位九肽库的构建及人Ⅳ型胶原酶特异结合肽的筛选

将人工合成的编码九肽的随机序列DNA片段克隆进丝状噬菌体表达载体FUSE5,经多次电击转化和表达,获得肽段与噬菌体pⅢ蛋白融合并展示在噬菌体表面的随机序列九肽表位肽库。库容量达10 10个克隆。以Ⅳ型胶原酶为靶蛋白,采用亲和纯化筛选模式,从中筛选出Ⅳ型胶原酶结合肽。进一步ELISA检测筛选出与Ⅳ型胶原酶特异结合的20个阳性克隆。序列分析发现一组肽含有WDXXD的共同序列,一组含有WVGXXR的共同序列。其中WDXXD的序列与Ⅳ型胶原酶单链抗体可变区序列同源。结果表明,多肽库是筛选蛋白特异结合肽的有力工具,表位九肽库的构建和筛选方法的建立为进一步应用筛选具有高亲和力的特异结合肽奠定了基础。     

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     

前列腺干细胞抗原(PSCA)的表达及其特异结合肽的筛选

通过反转录 PCR从人前列腺癌细胞中克隆了前列腺干细胞抗原 (PSCA)基因 ,在大肠杆菌中利用pQE30载体对截断型PSCA基因进行了可溶性表达。蛋白纯化后 ,利用噬菌体随机展示 12肽库筛选了PSCA蛋白的特异结合肽 ,通过与EGFP蛋白的耦联表达验证了结合肽的特异性。此特异结合肽的获得 ,为进一步研究针对PSCA的前列腺癌靶向免疫治疗奠定了基础     

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。     

应用噬菌体展示随机肽库淘筛mAb5H5识别的抗原表位

本文利用噬菌体随机9肽库探索汉滩病毒(HTNV)核衣壳蛋白(NP)B细胞抗原表位.以抗HTNV NP单克隆抗体(mAb)5H5作为筛选分子,生物淘洗噬菌体递呈的随机9肽库.阳性克隆经夹心ELISA、竞争ELISA鉴定后,随机挑取10个克隆,DNA测序,与HTNV 76～118株S基因进行同源性分析.结果显示筛选到的噬菌体能特异地与5H5结合,这种结合可被天然抗原所抑制.10个克隆的氨基酸序列相同,均为VRDAEEQYE,与76～118株NP氨基端的aa25～33一致.证实了该线性表位是mAb 5H5识别的表位,噬菌体肽库有助于病毒抗原表位的确定.     

抗人白细胞介素15中和抗体识别抗原表位的噬菌体肽库筛选与鉴定

目的:用噬菌体呈现随机七肽库筛选能与抗人白细胞介素15(h IL-15)中和抗体特异性结合的模拟抗原表位肽,并初步鉴定其免疫原性。方法:以抗h IL-15中和抗体为靶分子,用生物淘洗法从噬菌体呈现线性七肽库中筛选与之结合的噬菌体克隆,用噬菌体ELISA和竞争抑制ELISA鉴定阳性噬菌体克隆;化学合成筛选得到的多肽,并与匙孔血蓝蛋白(KLH)偶联免疫BALB/c小鼠,检测其免疫原性。结果:经过3轮体外筛选后随机挑取50个阳性噬菌体克隆,ELISA检测结果显示其中15个克隆与抗h IL-15抗体有较强的结合能力,DNA测序结果得到的结构相似群为MTPFWQK、MSPFNQK、MIPYWQK和MIPFHQK;竞争性ELISA结果显示4个序列均能与IL-15竞争性地结合抗IL-15单抗;小鼠免疫实验结果显示4组多肽均能诱导IL-15特异性免疫反应。结论:筛选得到能与抗h IL-15中和抗体特异性结合,且具有免疫原性的模拟抗原7肽序列,为进一步开发h IL-15相关的多肽疫苗提供了依据。     

大肠杆菌YggG与结合蛋白Era的相互作用研究

Era是细菌生长必须的一高度保守的GTPase。yggG是从大肠杆菌全基因组文库中钓取并克隆的Era结合蛋白基因,进一步的研究表明该基因在大肠杆菌中的表达与环境应激相关,提示yggG基因产物参与细菌的应激调控。为了阐明YggG蛋白与Era蛋白间的相互关系,利用所构建的双启动子表达载体pDH2-YggG-Ptac-Era在同一细胞中同时表达YggG与Era蛋白,并通过免疫共沉淀实验检测细菌裂解产物YggG与Era蛋白间的相互作用;在此基础上,构建并表达纯化了GST融合的Era蛋白氨基端截短肽和Era羧基端截短肽,通过GST Pull-down检测了Era不同功能区域与YggG蛋白间的相互作用。结果显示, Era/YggG 复合物仅存在于同时过表达Era和YggG蛋白的细菌细胞内,不诱导Era或者不诱导YggG蛋白过表达,均检测不到Era/YggG 复合物存在;纯化的GST不能Pull-down YggG蛋白,而纯化的GST融合的Era蛋白、Era氨基端截短肽及Era羧基端截短肽均可以Pull-down YggG蛋白;GST融合Era氨基端截短肽和GST融合的Era蛋白对YggG蛋白结合作用明显高于GST融合的Era蛋白羧基端截短肽。上述结果说明,YggG是一大肠杆菌Era结合蛋白,YggG与Era的氨基端和羧基端的结合活性存在差异。     

Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage.

Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a "secondary" library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.     

Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei

The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051^T possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95 kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051^T cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.     

Selection and properties for the recognition of P19 embryonic carcinoma stem cells

The P19 cell is a pluripotent stem cell of murine teratocarcinoma. When treated with retinoic acid, P19 cells can be differentiated along a neural cell lineage in culture. To isolate peptides that bind to the stem cell, we employed a phage display technology with undifferentiated P19 cells as the target. To reduce nonspecific binding of phages to the cell surface, the phage libraries were preadsorbed to the differentiated P19 cells before each selection on the undifferentiated P19 cells. After eight rounds of the selection, No. 28 phage displaying ALPSTSSQMPQL-peptide was isolated. Immunofluorescence analysis revealed that No. 28 phage selectively binds to the undifferentiated P19 cells but not to the differentiated P19 cells or SHSY cell line. The chemically synthesized peptide ALPSTSSQMPQL presented on the No. 28 phage efficiently inhibited the binding of No. 28 phage to the undifferentiated P19 cells. This result confirmed that No. 28 phage binding to the cell was mediated by the displayed peptide. The identified peptide may be targeted to a marker expressed on the stem cell and thus become a practical tool for the isolation of somatic stem cells.     

大分子量重组蛋白在丝状噬菌体表面的展示表达及其与小分子相互作用的初步研究

利用pHEN1KM13噬菌粒系统表达融合蛋白,进而确定大分子量重组蛋白在丝状噬菌体表达的部位及其表达后的生物活性。通过蛋白酶切处理前后噬菌体侵染细菌能力的变化快速地检测大分子蛋白质能否在噬菌体表面展示表达;比较了谷胱甘肽S转移酶及其与三个不同长度连接臂融合的外源蛋白在噬菌体表面的表达和组装,确定了不大于40kD的重组蛋白分子能展示表达在丝状噬菌体表面;并利用已知的小分子化合物与蛋白质的相互作用证明了组装在噬菌体表面的谷胱甘肽S转移酶重组蛋白仍保持其天然的结合活性,为利用噬菌体展示系统研究蛋白质与小分子化合物的相互作用建立了基础。     

Trichinella spiralis: characterization of phage-displayed specific epitopes and their protective immunity in BALB/c mice

Trichinellosis is a global zoonosis mainly caused by Trichinella spiralis. We have previously reported that a novel Ts87 gene from the cDNA library of adult T. spiralis was cloned and expressed in a prokaryotic expression system. Vaccination with recombinant Ts87 protein (rTs87) induced a muscle larvae burden reduction in BALB/c mice by 29% in response to T. spiralis infection. In the present study, we screened a random phage-displayed peptide library using monoclonal antibody 5A3 which recognized Ts87 protein. Four positive phage clones were selected to subcutaneously immunize BALB/c mice without adjuvant. Two phage clones could effectively stimulate specific antibodies against rTs87. Mice vaccinated with these two combined phage clones showed a 28.7% worm burden reduction as compared to the control group. Therefore, the identified phage clones displayed peptides representing specific epitopes of Ts87 protein and could be considered as potential vaccine candidates for T. spiralis.     

Production of an atrial natriuretic peptide variant that is specific for type A receptor.

Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein. These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution. This method also selected ANP variants with improved secretion expression in Escherichia coli. Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C. Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.     

Selection of a phage-displayed peptide recognized by monoclonal antibody directed blocking the site of hepatitis C virus E2 for human CD81

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). HCV envelope glycoprotein 2 (E2) most likely plays a pivotal role in binding to host cells by interacting with the hCD81 molecule. In this study, a phage-displayed peptide library was used to select small peptides with anti-hCD81 monoclonal antibody JS-81. The output/input ratio of phages increased about 91 fold after the third round of selection. Eight of the 30 phage clones selected from the phage library showed specific binding to the anti-hCD81 by enzyme linked immunosorbent assay (ELISA). Competitive inhibition test further demonstrated that HCV E2 could significantly inhibit the binding of a positive phage clone to anti-hCD81 JS-81. Exogenous small peptide ATWVCGPCT contained by the positive phage clones showed aligned with the hCD81 sequence from 153-161 by sequence analyses. These results suggest that the selected ATWVCGPCT is a novel hCD81-like small peptide, which can block the binding site of HCV E2 for hCD81. It may be of further application on development of antiviral agents targeting the stage of HCV entry.     

Design of a novel plasminogen activator based on the structure of hirudin

Using a phage library, seven peptide sequences with high affinity to human microplasminogen were obtained. Caseinolytic assay indicated that only the synthesized peptide P07 had slight fibrinolytic activity. To enhance its plasminogen activation ability, peptide P07 was fused into loop 32-35 of hirudin. In vitro assay demonstrated that this hirudin-like fusion protein can activate human plasminogen and retain the function of thrombin inhibition. Fusing the sequence ＂SPDASRL＂ into hirudin generated a plasminogen activation activity 100 times higher than peptide P07 in chromogenic and radial caseinolytic assay. This significant functional improvement might originate from a more specific active structure due to the hirudin scaffold.