Selection of trkB-binding peptides from a phage-displayed random peptide library

Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurode-generative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 mg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXfXXfXXC (X represents the random amino acids, f represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further devel-opment of small peptides mimicking BDNF activity.

Screening and Identification of a Novel Hepatocellular Carcinoma Cell Binding Peptide by Using a Phage Display Library

The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma （hHCC） cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide （WH-16） was synthesized. The competitive EL1SA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.

Identification and Characterization of Peptides Binding AgEG1 from a Phage Display Library

Endoglucanases are the main cellulolytic enzymes digestion as well as its good kinetic properties make it an attractive of Anoplophora glabripennis. Their high activities in cellulose target for development of cellulase inhibitors. In this study, random pepfide phage display technology was employed to identify peptides that bound the AgEG1, a member of endoglucanase isozymes. Phage clones with peptide LPPNPTK and XPP （X is residue T, L, A or H） motif frequently occurred in the selected phage population and showed a higher phage recovery than other clones. Peptide LPPNPTK was chemically synthesized and characterized tor its binding activities to AgEG1. The synthetic peptide exhibited high specificity for AgEG1. The peptide LPPNPTK has the potential to be developed into inhibitors of the endoglucanase of A. glabripennis.

A novel peptide, selected from phage display library of random peptides, can efficiently target into human breast cancer cell

To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 (9 aa) phage display library of random peptides. In multiple inde- pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate ex- tra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the peptide-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of pep- tide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by cocultur- ing them with other human tumor cell lines and normal cells. The nucleotide sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the peptide to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC (the shifted sequence of CASPSGALRSC), and after coculturing them with different cell lines, their targeting ca- pacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of peptides was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.

Screening of Peptide Inhibitors of TACE from a Phage Display Random 15-Peptide Library by Recombinant TACE Ectodomain

Tumor necrosis factor(TNF)-α-converting enzyme(TACE)is the major protease responsible for processing pro-TNF-αfrom membrane-anchored precursors to secreted TNF-α.In the present study,a 15-peptide library was used to identify potential TACE antagonists.To obtain the recombinant TACE ectodomain and to use it as a selective molecule for the screening of peptide inhibitors of TACE,cDNA coding for the catalytic domain(T800)and full-length ectodomain(T1300)of TACE were amplified by reverse transcription–polymerase chain reaction.The expression plasmid were constructed by inserting T800/T1300 into plasmid pET-28a/c respectively and were transformed into Escherichia coli BL21(DE3).Sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDSPAGE)andWestern blot analysis revealed that T800/T1300 were highly expressed in the form of an inclusion body induced by isopropylthiogalactoside.After Ni2+–NTA resin affinity chromatography,the purity of the recombinant T800/T1300 protein was more than 90%.T800 and T1300 proteins were used in the screening of T800/T1300-binding peptides from a phage display random 15-peptide library.After four rounds of biopanning,the positive phage clones were analyzed by enzyme-linked immunosorbent assay,competitive inhibition assay(ELESA),and DNA sequencing.A common amino acid sequence(TRWLVYFS RPYLVAT)was confirmed and synthesized.A synthetic peptide was shown to bind to TACE and to inhibit TNF-αrelease from lipopolysaccharide(LPS)-stimulated human peripheral blood mononuclear cells(PBMC)by up to 60.3%.Fluorescence-activated cell sorter(FACS)analysis revealed that the peptide mediated the accumulation of TNF-αon an LPS-stimulated PBMC surface.These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and that the deduced motif might be applied to the molecular design of anti-inflammatory drugs.

日本血吸虫模拟虫卵抗原表位的筛选和鉴定

目的 通过噬菌体肽文库技术探索可用于诊断日本血吸虫病的模拟虫卵抗原。 方法 用日本血吸虫虫卵单克隆抗体 6 B12作“诱饵”,采用生物淘金法从噬菌体 15 -肽文库中经过 3轮筛选 ,有效地富集与 6 B12有亲和力的噬菌体肽克隆。随后采用 EL ISA、竞争 EL ISA、Western blotting等检测方法 ,从 4 0 0个单克隆化的噬菌体株中得到 13株与 6 B12有特异性、高亲和力反应的噬菌体克隆。 结果 对噬菌体所携带的外源 DNA片段序列测序结果表明 ,13株噬菌体所携带的外源多肽序列完全相同。该噬菌体多肽抗原包被酶标板经 EL ISA方法对血吸虫病患者及健康人血清Ig G抗体检测结果表明 ,两者有显著性差异。

从HCV核心蛋白噬菌体随机展示肽库中筛选抗原表位

目的探讨病毒抗原序列研究的新方法。方法应用噬菌体展示技术，筛选ＨＣＶ核心蛋白的抗原序列。用抗－ＨＣＶ核心抗体单独阳性的血清，对巳构建的ＨＣＶ核心蛋白噬菌体随机展示肽库进行４轮筛选，检测筛选阳性的克隆数、插入阳性率和杂交阳性率来评价筛选的效果。测定和分析７个杂交阳性克隆的ＤＮＡ序列。结果所测定的７个序列中，６个为ＨＣＶ核心蛋白序列，其中５个被正确地展示于噬菌体表面，１个可能被正确展示，这些序列均含有重要的ＨＣＶ核心抗原表位。另１个为大肠杆菌ｎｒｆａ基因。结论噬菌体展示技术完全可应用于病毒蛋白抗原序列的筛选，并具有简便、快速、准确的优点。

HIV蛋白质优势B细胞抗原表位的筛选、重组表达与抗原性鉴定

设计了一种新的病原体蛋白质B细胞抗原表位的筛选和重组表达方法。不须使用抗原 ,而通过交替用病人血清IgG抗体“淘洗”(biopanning)随机肽库和用正常人血清IgG反向吸附 ,来获得特异抗原表位资料。用HIV病人血清的IgG抗体淘洗噬菌体递呈随机十二肽库 ,再以正常人IgG抗体吸附 ,筛选到了能和HIV病人血清发生特异反应的噬菌体克隆 ,经ELISA、DNA测序等 ,成功地筛选出了位于HIVgp41外膜蛋白、高亲和力、构型特异的优势B细胞抗原表位 ( 60 2 GCSGKLICTTNV61 3)。用大肠杆菌硫氧还蛋白作为骨架 ,在其活性部位以“内融合”形式重组表达了该抗原表位 ,纯化的重组蛋白具有良好的抗原性 ,能与HIV( + )IgG抗体及艾滋病人血清呈特异反应 ,表明本技术路线可以有效地进行HIV蛋白质的B细胞抗原表位筛选和重组表达。此方法也可移植于其它病原微生物抗原或自身抗原的表位研究 ,继而为基于抗原表位水平的特异诊断试剂的研制、疫苗的设计提供依据。

HCV核心蛋白噬菌体随机展示肽库的构建

目的 :为了探讨噬菌体展示技术在筛选和鉴定病毒抗原表位研究中的应用前景。 方法 :利用随机引物 PCR法以HCV核心基因为模板合成随机 DNA片段 ,再经特定引物二次扩增后获得高丰度的 HCV核心蛋白随机 DNA片段 ,将该片段插入噬菌粒载体 p CANTAB5 X的 Xba 位点 ,转化大肠杆菌 TG1,辅助噬菌体拯救 ,建立噬菌体随机肽展示文库。结果 :所构建的随机文库含 1.2 3× 10 5个不同克隆 ,库容噬菌体滴度为 2× 10 1 2 TU/ m l(转导单位 / ml) ,PCR法检测插入阳性为 2 8.1% ,核酸杂交证实 40 %的克隆为 HCV核心基因阳性克隆。 结论 :所建的随机文库有足够大的库容和较好的多样性 ,可以满足HCV C抗原表位的筛选。

运用噬菌体展示随机肽库筛选与内毒素结合的高亲和性多肽

目的从噬菌体展示随机肽库中筛选与内毒素结合的多肽序列,并进行鉴定。方法以内毒素脂多糖(lipopolysaccharide, LPS)为靶分子对噬菌体展示随机十二肽库进行4轮亲和筛选,获得与LPS结合的噬菌体克隆,应用结合实验和克隆斑抑制实验进一步确证。挑选结合力强的克隆进行DNA测序,推导出呈现的多肽序列,应用生物信息学软件进行多肽序列分析和同源性分析。结果经4轮亲和筛选从噬菌体展示随机十二肽库中筛选获得了86个克隆,挑选12个结合力强的克隆进行DNA序列测序及生物信息学分析,结合本项目组的噬菌体展示随机七肽库筛选结果推导出呈现的多肽序列为HWQWPHWSPPP(命名为P11肽)。检索相关数据库发现此肽序列未被申请专利,体内约908种蛋白与其结构相匹配,其中包含有与LPS相互作用的位点。结论通过对噬菌体展示随机肽库的淘选,获得与LPS结合的高亲和性多肽,为进一步以这些多肽为先导物进行定向进化研究提供了实验依据和结构基础。

脂多糖保守表位模拟肽的筛选与鉴定

用针对脂多糖保守表位的单抗 2B4对噬菌体随机 12肽库进行亲和筛选 ,通过噬菌体ELISA实验及脂多糖(LPS)竞争抑制实验鉴定阳性克隆 .经三轮筛选后 ,与抗体结合的噬菌体得到明显富集 ,噬菌体ELISA结果显示 ,阳性率达 80 % .将其中 12个阳性噬菌体克隆做鼠伤寒杆菌和大肠杆菌LPS竞争抑制实验 ,抑制作用非常明显 ,有良好的剂量依赖关系 ,证明这 12个克隆与LPS具相似表位 .DNA测序并推导噬菌体展示肽的氨基酸序列为 ,GPPQWFFSQPQL (5 / 12 ,41 7% ) ,LPQYFWNTATTA (3/ 12 ,2 5 % ) ,FPQNHWNVPWAT (2 / 12 ,16 6 % ) ,HSQSFWNAPLAM和AHPWTHGYFPPL (1/ 12 ,8 3 % ) .实验结果表明 ,用 2B4抗体筛选到的噬菌体短肽克隆可模拟保守表位 ,即脂多糖的模拟肽 (位 ) .

从噬菌体随机肽库中筛选IL-6抗体的识别表位

目的 :从噬菌体随机 6肽文库中筛选IL 6抗体的识别表位。方法 :利用具有IL 6中和活性的单抗C2 2 0筛选呈现于丝状噬菌体表面P3蛋白的随机 6肽文库。结果 :从噬菌体随机 6肽文库中筛选出 36株可与单抗特异结合的噬菌体克隆 ,其中多数克隆呈现核心序列SWLR ,该序列与IL 6的序列具有一定同源性。竞争结合实验和Western印迹分析 ,带有SWFNLR和HSWLRY小肽的 2株噬菌体克隆可与IL 6竞争结合单抗C2 2 0。结论 :SWFNLR和HSWLRY是IL 6单抗C2 2 0特异识别的表位。

HA结合肽的筛选与序列分析

利用噬菌体随机十二肽库对羟基磷灰石进行结合肽筛选,经4轮生物淘洗和选择、噬菌体扩增和DNA测序,获得一组多肽序列,其中含有较高比例的脯氨酸、亮氨酸、谷氨酸、谷氨酰胺以及疏水氨基酸.经Blast分析、CLUSTAL W多重序列比对推得羟基磷灰石结合肽可能的结合基序为VLPP、LP(X)6PL/X、PP(X)4～6P,(X为任意氨基酸).认为脯氨酸等氨基酸的高比例特性以及结合基序特征可以很好地解释羟基磷灰石的生物可利用性和安全性.在生物体中未发现结合肽有价值的相似DNA编码序列和多肽、蛋白质序列.噬菌体单克隆反筛功能测定进一步确定了相关序列对羟基磷灰石的结合能力.

噬菌体表达短肽模拟血吸虫致弱尾蚴特异性抗原表位的研究

目的 从噬菌体随机肽库中筛选模拟血吸虫致弱尾蚴特异性抗原表位的噬菌体克隆 ,并探讨其免疫保护效果。方法 用紫外线致弱尾蚴免疫兔血清IgG筛选以融合蛋白形式在丝状噬菌体外壳蛋白Ⅲ表达的随机 7肽库 ,三轮免疫筛选后获得的噬菌体克隆经皮下免疫小鼠 3次 ,攻击感染 45d后剖杀 ,观察减虫和减卵效果。结果 经三轮筛选 ,特异性结合的噬菌体富集增加了 10 0多倍 ,随机挑取 2 4个噬菌体克隆经ELISA测定 ,有 2 2个克隆能与致弱尾蚴免疫兔血清IgG特异性反应。混合噬菌体克隆免疫小鼠试验的减虫率与减卵率分别为 33.5 7%和 5 6 .0 7% (P均 <0 .0 0 1)。结论 采用噬菌体随机肽库技术可获得模拟致弱尾蚴特异性抗原表位的短肽分子 。

一种稳定、易于操作的噬菌体随机肽库载体的构建

目的：拟将噬菌粒ｐＣＯＭＢ３改建为更为稳定、高拷贝且易于操作的噬菌体随机肽库载体。方法：对常用噬菌体抗体库呈现载体ｐＣＯＭＢ３序列进行点矩阵作图分析（Ｄｏｔ－Ｐｌｏｔ分析），切除其中重复区，纠正其多代培养后自删除特性。原克隆位点６３ｂｐ的填充序列增加到１４３５ｂｐ以便于酶切鉴定和电泳回收。结果：构建了命名为ｐＴＭＢ２的噬菌粒载体。结论：ｐＴＭＢ２的噬菌粒载体更为稳定且易于操作。

单纯疱疹病毒糖蛋白模拟位的研究

目的为了充分认识单纯疱疹病毒(HSV)糖蛋白的抗原表位，寻找HSV多组分疫苗和新型基因工程疫苗更有效的小片段短肽作为该病毒疫苗的备选免疫原。方法利用有动物保护活性的抗HSV糖蛋白C（gC）单克隆抗体（McAb）1A12淘筛噬菌体随机12肽库，经4轮筛选后进行ELISA检测，随机挑取14个阳性克隆进行序列测定,序列比较后得到保守序列，做ELISA阻断实验进一步鉴定筛选结果并与相关天然蛋白HSV gC进行序列比较。结果获得保守序列-SG(L)RHII-和其侧翼辅助序列-AK-、-VW-，其与天然相关蛋白HSV gC 114～116位氨基酸十分相似。携有此短肽的噬菌体可以与McAb特异结合，并可部分阻断抗体与HSV囊膜抗原的反应。结论所筛选到的保守序列可模拟McAb针对的抗原表位，可能是HSV的替代抗原。这一研究方法有望使短肽替代庞大的糖蛋白全长氨基酸序列，为研制更有效及更广谱的基因工程疫苗提供依据，也使研制基于抗原表位水平的特异诊断试剂成为可能。

噬菌体展示短肽模拟脂多糖结合蛋白抗炎功能位点的研究

目的 应用噬菌体随机 12肽库筛选脂多糖结合蛋白 (lipopolysaccharidebindingprotein ,LBP)具有抗炎作用的肽序列。方法 以LPS为目标分子 ,对噬菌体十二肽库进行亲合筛选 ,4轮筛选后 ,检测随机挑取的噬菌体克隆的结合活性和抑制活性 ,对可与LBP竞争性结合LPS的噬菌体克隆行细胞因子生成抑制实验和LPS内化抑制实验 ,对获得的阳性克隆进行DNA序列测定 ,推导外源肽氨基酸序列 ,并与LBP序列比较 ,确定LBP的抗炎功能位点。结果 随机挑取的 10 0个噬菌体克隆中 16个可与LBP竞争性结合LPS ,其中 7个噬菌体克隆对LBP的增敏LPS刺激PBMC分泌TNF α功能无作用 ,对这 7个克隆进行LPS内化抑制实验 ,其中 3个克隆可以抑制LBP增强LPS内化作用。测序发现这 3个阳性克隆融合多肽的序列均为FHTRWNYWPYLH ,与LBP一级结构氨基酸序列同源性低。结论 该

噬菌体展示免疫球蛋白结合分子单结构域随机组合文库的构建

目的:构建由免疫球蛋白结合分子单结构域随机组合的噬菌体展示文库,为应用各种类型免疫球蛋白对该文库进行体外分子进化筛选研究打下基础。方法:PCR扩增制备分别编码Protein A 的A、D结构域、Protein G的B2 结构域和Pro tein L的B3结构域的序列片段,片段两端引入XbaⅠ酶切位点,3′端引入分别编码0、1、2、3个氨基酸大小随机连接肽的序列,并克隆于pMD 18T载体构建重组质粒。以上述16种重组质粒为模板,PCR扩增分别制备各种单结构域片段及两端部分T载体序列,XbaⅠ酶切,各种酶切片段混合后连接,并克隆于噬菌体展示载体pCANTAB5S,构建噬菌体展示免疫球蛋白结合分子单结构域随机组合文库,挑取单克隆进行序列测定,评价文库的随机性。结果:噬菌体展示免疫球蛋白结合分子单结构域随机组合文库库容量为2×107,滴度为1.3×1011;文库中含77%以上的阳性克隆,其中由2个以上单结构域组成的阳性克隆占24%;序列分析显示组合文库包含上述4种单结构域序列,连接方式符合随机连接的特点,随机连接肽的编码核酸也呈随机分布。结论:成功构建了免疫球蛋白结合分子单结构域随机组合文库,该文库库容、多样性和随机性完全可满足进行体外分子进化研究的要求。

法氏囊活性五肽BP5特异性结合肽的筛选及鉴定

为鉴定与具有免疫调节功能和抗肿瘤潜能的法氏囊活性五肽(BP5)特异性结合的多肽,本研究利用噬菌体展示技术,以BP5-BSA为靶分子,对噬菌体随机12肽库进行4轮亲和筛选,结合ELISA鉴定和竞争抑制试验,筛选出3个能够特异性与BP5结合的噬菌体阳性克隆。测序结果显示,其编码的12肽序列分别为:PINMQTLNCMAA(P2-12)、GCTLNPMSDLLG(P6-12)和MSSTLNGMLNSL(P7-12),其核心基序为TLNXM。通过人工合成P2-12、P6-12、P7-12多肽,并采用MTT法检测其对BP5抗肿瘤细胞增殖能力的影响,结果显示,P2-12和P7-12肽在0.2μg/m L^20μg/m L浓度下,P6-12肽在2μg/m L^20μg/m L浓度下均能够抑制BP5抗小鼠WEHI-231 B淋巴瘤细胞增殖作用,其中P2-12在20μg/m L浓度下抑制作用最为显著(p<0.01)。此外,p53荧光素酶活性检测结果显示,这3种BP5结合肽在实验浓度下均能够下调BP5促p53基因转录的活性。以上结果表明,这3种BP5特异性结合肽具有下调BP5抗肿瘤活性。本研究为进一步研究BP5抗肿瘤细胞增殖作用的机制提供了相关的实验依据。

NF-κB p65亚基DNA结合域相互作用多肽的筛选及功能鉴定

目的以NF-κB p65亚基DNA结合域为诱饵蛋白,筛选能够与其发生相互作用的多肽,并鉴定这些多肽对NF-κB DNA结合活性的抑制效应。方法首先利用PCR搭桥扩增方法获取NF-κB p65亚基DNA结合域的基因片段,后将其克隆到酵母双杂交pGBKT7DNA-BD载体上,与GAL4DNA-BD融合,形成GAL4DNA-BD/p65BD融合蛋白;再以GAL4DNA-BD/p65BD融合蛋白为诱饵蛋白行酵母双杂交实验,自16肽随机肽库中筛选与NF-κB p65亚基DNA结合域相互作用的多肽;最后选择性人工合成筛选获得的多肽,并进行EMSA实验检测相互作用多肽对NF-κB DNA结合活性的抑制效应。结果扩增获得了NF-κB p65亚基DNA结合域的基因片段,并成功构建pGBKT7DNA-BD/p65BD载体。自1.28×107个酵母转化子中最终筛选获得5个阳性克隆,测序结果和同源氨基酸序列比对表明5个阳性多肽为p65BD新型相互作用多肽,其中4条多肽拥有一个共同的基序。EMSA实验结果表明合成的两条多肽对NF-κB的DNA结合活性均具有一定的抑制效应。结论经初筛、复选和假阳性鉴定,自16肽库中获得5个能与NF-κB p65亚基发生相互作用的新型多肽,并已证实其中两条多肽对NF-κB的DNA结合活性具有抑制效应。