三阴性乳腺癌原代细胞的培养及其特异性结合肽的筛选

目的利用噬菌体肽库筛选技术获得与三阴性乳腺癌原代细胞特异性结合的多肽,为探索三阴性乳腺癌的治疗靶点提供试验支持。方法从三阴性乳腺癌新鲜组织分离、培养原代癌细胞并以其为靶细胞,以hs578bst人正常乳腺细胞为吸附细胞,对噬菌体随机十二肽库进行三轮减性筛选;随机挑取15株富集后的噬菌体单克隆,ELISA及DAB染色鉴定噬菌体克隆的亲和力及特异性,并对阳性噬菌体单克隆测序。结果经过3轮筛选,噬菌体克隆得以明显富集,随机挑取的15株噬菌体单克隆中有11株为阳性,ELISA及DAB鉴定发现,5号噬菌体(phage-5)对靶细胞亲和力及特异性最强,经DNA测序和推导,其多肽序列为PHETLTSFVRRG。结论从噬菌体随机十二肽库中成功筛选出与三阴性乳腺癌原代细胞特异性结合的多肽,该多肽可能作为三阴性乳腺癌靶向治疗药物的载体。     

用噬菌体展示技术筛选喉癌细胞靶向肽的研究

目的筛选人源喉癌Hep-2细胞株特异结合的短肽,作为喉癌靶向治疗的载体。方法体外培养Hep-2细胞株作为靶细胞,人正常喉黏膜上皮细胞为吸附细胞;用噬菌体展示十二肽库进行3轮差减筛选,随机挑取10个噬菌体克隆进行测序;采用酶联免疫吸附（enzyme linked immunosorbent assay,ELISA）法鉴定噬菌体与Hep-2细胞的结合活性;通过免疫荧光鉴定喉癌细胞特异性结合肽（F2）噬菌体阳性克隆与喉癌细胞结合的特异性。结果经过3轮筛选后,噬菌体在靶细胞Hep-2上出现明显富集;ELISA分析鉴定显示5个阳性克隆能与Hep-2细胞特异结合,其中F2噬菌体克隆对喉癌细胞的结合靶向性明显高于对照细胞（P〈0.05）;免疫荧光显色显示,F2能特异性地与喉癌细胞结合。结论利用噬菌体展示肽库技术,可以成功筛选到F2,其可能成为喉癌靶向治疗的载体。     

噬菌体环7肽库筛选胰岛素受体亲和肽

目的:筛选胰岛素受体高亲和肽作为肝细胞癌潜在靶向载体。方法:用噬菌体随机环七肽库,与鼠肝胰岛素受体亲和、筛选与扩增。经过三个循环的筛选,测定阳性噬菌体与胰岛素受体的亲和力,测定亲和力高的阳性噬菌体克隆插入DNA序列,推断出与噬菌体外壳gⅢ蛋白融合的短肽氨基酸残基序列。分析亲和短肽结构特征,固相法选择性人工合成多肽配基TK1,测定多肽TK1与胰岛素受体的亲和力。结果:经过三轮筛选,选择12个阳性克隆测序,11个克隆插入短肽是TK(CysGlnSerLysHisTrpArgHisCys),合成肽TK1(CysGlnSerLysHisTrpArgHisCysTyr)与胰岛素受体有较高亲和力,Kd值为4.31×108L/M。结论:噬菌体肽库筛选小鼠肝胰岛素受体获得了两种多肽配基,多肽TK1与胰岛素受体有较高亲和力,可望作为肝癌靶向多肽,值得进一步研究。     

筛选及鉴定IgG型单克隆抗-D噬菌体克隆的实验研究

目的利用随机噬菌体12肽库筛选出血型D抗原的模拟多肽并验证。方法采用Ig G型单克隆抗-D筛选噬菌体随机12肽库,测定每轮筛选后洗脱物与抗-Rh D的结合情况。对经特异性筛选得到的阳性噬菌体克隆进行DNA序列测定,将推导出的12肽氨基酸序列进行同源性比较,通过凝集抑制试验验证阳性噬菌体与Ig G型单克隆抗-D的反应。结果随着筛选轮次增加,噬菌体洗脱物与抗-Rh D的结合力逐渐增强。经过4轮淘洗后,得到8个亲和力较强且具有较高重合率的保守区域的12肽序列。凝集抑制实验表明具有相同氨基酸序列的阳性噬菌体对Ig G型单克隆抗-D与Rh D阳性红细胞的凝集反应具有抑制作用。结论随机噬菌体12肽库筛选得到的血型D抗原模拟多肽,为Rh D结构与功能研究及研制针对Rh D新生儿溶血病的新型诊断抗原、制备特异疫苗以及探索新的治疗方法提供实验基础。     

人Rh(D)血型抗原模拟表位的筛选和鉴定

目的 从噬菌体随机肽库中筛选人Rh(D)血型抗原模拟表位,并对其免疫学活性进行鉴定.方法 以抗Rh(D)单克隆抗体作为固相筛选分子,对噬菌体随机十二肽库进行3轮"吸附-洗脱-扩增"筛选过程,随机挑选35个克隆,经噬菌体ELISA和交叉反应试验,确定阳性克隆,再对这些克隆进行DNA序列测定和抗体竞争抑制试验,以获取人Rh(D)血型抗原模拟表位.然后采用蛋白质免疫印迹法(Western blot)对所获噬菌体进行鉴定和抗原性分析.结果 经过3轮淘洗后,噬菌体得到富集,获得11个阳性克隆.DNA序列测定和竞争抑制实验结果表明,这11个克隆噬菌体所展示的外源多肽中有7个克隆氨基酸序列含有相同的色氨酸(W)、脯氨酸(P)和谷氨酰胺(Q)结构(-WP-Q-),且均具有40%以上的抑制率;其余4个克隆没有共性,抑制率较低,可能为非特异性结合.Western blot分析表明,被选定的噬菌体能被抗Rh(D)血清特异识别,具有类似于Rh(D)蛋白的抗原性.结论 利用抗Rh(D)单克隆抗体筛选噬菌体随机肽库,成功获得含有(-WP-Q-)结构的Rh(D)抗原模拟表位,为进一步探讨Rh(D)新生儿溶血病的发病机制和疫苗的研究奠定基础.     

抗TNF-α单克隆抗体对噬菌体随机12肽库的筛选

[目的]试从噬菌体随机多肽库中筛选出能与肿瘤坏死因子-α(TNF-α)特异性结合的短肽分子。[方法]采用抗TNF-α单克隆抗体作为配基，免疫亲和筛选以融合蛋白形式在丝状噬菌体M13外壳蛋白Ⅲ表达的随机12肽文库，按“吸附一洗脱一扩增”的淘洗过程，经过3轮免疫筛选后，随机挑取阳性噬菌体克隆用ELISA及斑点ELISA检测其特异性。[结果]经过3轮免疫筛选，特异性结合的阳性噬菌体克隆富集增加近100倍，第3轮筛选后随机挑选7个阳性噬菌体克隆经ELISA和斑点ELISA检测，有5个克隆能与抗TNF-α特异性结合。[结论]噬菌体随机肽库技术可以应用于分析、鉴定抗TNF-α的表位，继而为获得亚单位表位水平的诊断试剂和抗TNF-α的短肽疫苗提供依据。     

噬菌体展示技术筛选乙型肝炎病毒X蛋白相互作用蛋白

目的利用T7噬菌体展示技术筛选与乙型肝炎病毒X蛋白相互作用的蛋白。方法构建X蛋白的原核表达载体,表达并纯化X蛋白,以X蛋白作为靶蛋白,应用T7噬菌体展示技术对人肝细胞cDNA文库进行筛选,对筛选到的阳性克隆进行DNA序列分析及同源性研究。结果表达并纯化了X蛋白,经过4轮筛选后,选择52个阳性克隆,采用T7特异性引物扩增插入片段,回收PCR产物进行测序。经过同源性分析,确定了7个与乙型肝炎X蛋白相互作用的蛋白。结论 T7噬菌体展示筛选是1种快速、简单的筛选蛋白质相互作用的工具,筛选的与X蛋白相互作用蛋白为进一步研究乙型肝炎病毒的致病机制奠定了基础。     

膀胱癌细胞系T24特异性导向小分子多肽的筛选

目的膀胱原位癌和微小转移灶的诊断和治疗目前尚未取得令人满意的效果,寻找高特异性和高结合力的肿瘤导向多肽已成为提高诊断和治疗效率的研究热点。本研究旨在利用噬菌体随机肽库获得与膀胱尿路上皮细胞癌细胞株T24特异结合的小分子多肽序列,以期作为膀胱癌靶向诊断和治疗的导向载体。方法利用噬菌体随机7肽库对肿瘤细胞进行4轮全细胞筛选,分析筛选后单克隆对膀胱癌细胞的特异性结合能力。提取单克隆DNA并测序,得出多肽序列进行对比分析。结果经过4轮筛选后所挑选的20个单克隆均显示与膀胱癌细胞有较高的特异性结合,并测序得到四条重复性高的多肽序列（-SNARGTE-、-VSEKNRQ-、-DSIYNAR-、-DWS-GACS-）。结论通过噬菌体随机肽库对膀胱癌细胞进行全细胞筛选得到的噬菌体多肽能与膀胱癌细胞T24特异性结合,初步可作为膀胱癌靶向诊断和导向药物研究的载体。     

晚期氧化蛋白产物模拟表位的初步研究

目的利用噬菌体展示肽库筛选技术获得模拟人晚期氧化蛋白产物（advanced oxidation protein products,AOPP）表位的序列,探索该表位的分布与表达。方法以抗晚期氧化蛋白产物多克隆抗体为靶,对噬菌体随机12肽库进行免疫亲和筛选,ELISA鉴定阳性噬菌体克隆特异性;对阳性克隆进行DNA测序并推导其氨基酸序列,利用生物信息学技术检索其分布与相似性。结果经3轮亲和筛选获得18株与靶分子特异结合的阳性噬菌体克隆,阳性率为62.07%。竞争ELISA结果表明,AOPP可有效抑制阳性噬菌体克隆与抗体结合,其中对No.6克隆的IC50达到0.2ug/ml,提示其能模拟AOPP表位。经DNA测序并推导氨基酸序列,得到LXDMLXD保守序列（X为任意氨基酸）;发现该序列不存在于正常人与哺乳动物的蛋白分子,但与某些真菌及寄生虫蛋白分子有同源性。结论通过噬菌体肽库筛选技术获得模拟AOPP表位并具良好抗原性的短肽序列;证实该结构不存在于正常人与哺乳动物蛋白分子,正如AOPP并非正常组织蛋白。     

应用噬菌体展示技术筛选血型A抗原表位的模拟多肽

目的 筛选血型A抗原表位的模拟多肽,探讨其在血型A抗体检测中的应用价值.方法 用血型A抗原特异性单克隆抗体NaM87-1F6从噬菌体随机12肽库中筛选能与之结合的噬菌体展示肽.采用噬菌体ELISA、噬菌体微筛选试验鉴定噬菌体与抗体的结合力.DNA测定并推断特异性噬菌俸克隆展示的多肽序列.凝集抑制试验检测单克隆噬菌体对抗体凝集红细胞的抑制作用,ABO-ELISA初步分析噬菌体展示多肽检测血型抗体的效能.结果 经筛选和鉴定后得到7个阳性克隆,其中6个展示多肽序列为EYWYCGMNRTGC(C5),1个为QIWYERTLPFTF(C17).凝集抑制试验提示2个噬菌体展示多肽可以特异性抑制NaM87-1F6对A型红细胞的凝集作用.基于噬菌体多肽C5、C17的ABO-ELISA检测血浆抗A抗体试验的受试者操作特性曲线下面积分别为0.889(P=0.000)和O.751(P=0.000),ABO-ELISA值与凝集试验抗体滴度间的Spearman相关系数分别为0.743(P＜0.01)和0.664(P＜0.01).基于C5的ABO-ELISA在临界值为0.300时其敏感度、特异度分别为82.2%和83.3%;基于C17的ABO-ELISA在临界值为0.250时其敏感度、特异度分别为68.9%和63.3%.结论 多肽EYWYCGMNRTGC,QIWYERTLPFTF可以模拟血型A抗原的表位.基于这些多肽的ABO-ELISA方法具有检测血型A抗原特异性抗体的潜能.     

人Rh(D)血型抗原模拟表位筛选及鉴定的临床意义

【目的】探讨人Rh(D)血型抗原模拟表位筛选及鉴定的临床意义。【方法】以抗Rh(D)单克隆抗体作为固相筛选分子,随机选取35个进行克隆,经噬菌体酶联免疫吸附测定法(ELISA)及交叉试验,确定阳性克隆,采用蛋白石免疫印迹法(Western blot)对噬菌体行抗原性分析。【结果】DNA序列测序及竞争抑制实验在11个克隆的噬菌体所展示的外源多肽中有4个克隆无共性,可能是非特异性结合。7个具有克隆氨基酸序列均含有相同的脯氨酸(P)、色氨酸(W)、谷氨酸(Q)结构(-WP-Q-),超过40%的抑制率。Western blot分析表明,被选定的噬菌体能被抗Rh(D)血清特异识别,抗原性与Rh(D)蛋白相类似。【结论】利用抗Rh(D)单克隆抗体筛选噬菌体随机肽库,可获得具有(-WP-Q-)结构的Rh(D)抗原模拟表位。     

Identification of a VWF peptide antagonist that blocks platelet adhesion under high shear conditions by selectively inhibiting the VWF-collagen interaction

Summary.  Background : Because the collagen-VWF-GPIb/IX/V axis plays an important role in thrombus formation, it represents a promising target for development of new antithrombotic agents. Objectives : We used phage display to identify potential small peptides that interfere with the VWF-collagen binding and might serve as lead products for the development of possible oral antithrombotic compounds. Methods : A random linear heptamer peptide library was used to select VWF-binding peptides. Results : We identified a phage clone, displaying the YDPWTPS sequence, further referred to as L7-phage, that bound to VWF in a specific and a dose-dependent manner. This L7-phage specifically inhibited the VWF-collagen interaction under both static and flow conditions. Epitope mapping using deletion mutants of VWF revealed that the L7-phage does not bind to the known collagen-binding A3 domain within VWF, but to the more carboxyterminal situated C domain. This inhibition was not due to steric hindrance of the A3 domain-collagen interaction by the L7-phage. Indeed, a tetrabranched multi-antigen peptide (MAP) presenting four copies of the peptide, but not the scrambled MAP, also inhibited VWF-collagen interaction under conditions of high shear stress at a concentration of 148 nmol L⁻¹. Conclusions : Based on these results, we conclude that we have identified the first peptide antagonist that binds to the VWF C domain and by this specifically inhibits the VWF binding to collagen, suppressing platelet adhesion and aggregation under high shear conditions. As a consequence, this peptide and its future derivates are potentially interesting antithrombotic agents.     

Intracellular expression of Peptide fusions for demonstration of protein essentiality in bacteria

We describe a "protein knockout" technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.     

急性淋巴细胞白血病单链抗体(scFv)的筛选与鉴定

目的 筛选急性淋巴细胞白血病病人单链可变区scFv抗体,为进一步表达并得到其特异性强的抗体片段创造条件。方法 实验利用初诊急性淋巴细胞性白血病病人血清为包被抗原,采用噬菌体表面展示技术,从半合成的人源噬菌体抗体库中筛选其特异性噬菌体抗体,首先把靶抗原包被于免疫平板后,加入噬菌体抗体库,这样能与靶抗原特异性结合的噬菌抗体就被固定在免疫平板上,不能特异结合的噬菌体则被漂洗掉; 将特异结合的噬菌体洗脱下来,侵染大肠埃希菌,就可以得到含特异抗体基因的噬菌粒。结果 经过3轮“吸附-洗脱-扩增”筛选过程,获得抗原特异性较强的白血病病人可变区噬菌体抗体片段并鉴定。结论 获得了一株亲和性较好的抗体片段,为下一步片段表达、鉴定及临床应用研究创造了条件。     

Screening serum biomarkers for early primary hepatocellular carcinoma using a phage display technique

Hepatocellular carcinoma (HCC) occurs mainly in chronically diseased livers following hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Early detection and diagnosis of HCC would be of great clinical benefit. In this study, we used a random phage display peptide library and sera from early-stage primary HCC patients (n = 30) to screen potential serum biomarkers for early primary HCC. Age- and sex-matched patients with HBV and/or HCV infection were used as controls. In the screening phase, 19 out of 20 randomly selected phage clones exhibited specific reaction with purified sera IgG from early primary HCC patients, among them 14 coming from the same phage clone with inserted peptidesequence RGWCRPLPKGEG (named HC1). In the validation phase, phage ELISA results showed that the positive reaction rate of the HC1 phage clone was 91.4% with the early HCC group (n = 70), significantly higher than that with the HBV infection group (20.0%) (n = 70), the HCV infection group (12.9%) (n = 70), the HBV + HCV infection group (24.3%) (n = 70), the cirrhosis group (17.1%) (n = 70), and the healthy control group (10.0%) (n = 70). In conclusion, the HC1 mimic peptide showed high diagnostic validity for early primary HCC, and thereby could be a candidate serum biomarker for early primary HCC.     

Donor screening for antibody to hepatitis B core antigen and hepatitis B virus infection in transfusion recipients

BACKGROUND: Testing for antibody to hepatitis B core antigen (anti-HBc) as a surrogate for hepatitis C viremia is no longer needed for blood donor screening. Currently, the important question is how much its use supplements hepatitis B surface antigen (HBsAg) donor screening in preventing transfusion-transmitted hepatitis B virus (HBV) infection. STUDY DESIGN AND METHODS: In a study conducted in the 1970s, 64 blood donors were associated with 15 cases of HBV (1.0%) in 1533 transfusion recipients. Sera from 61 donors at donation and 29 follow-up visits were available for present-day assays for HBsAg, HBV DNA, anti-HBc, and antibody to HBsAg (anti-HBs). RESULTS: HBsAg was found in four previously negative blood donors; HBV DNA was limited to three of these four. Anti-HBc was detected in six HBsAg-negative donors. Two other donors were negative in all assays at donation, but positive for anti- HBc and anti-HBs 2 to 4 months later. The remaining donors were negative for all HBV markers, which left five recipient cases unexplained. No HBV transmission was observed when anti-HBs sample-to- negative control values were > or = 10. CONCLUSION: Some 33 to 50 percent of cases of hepatitis B that could be transmitted by transfusion of blood from HBsAg-negative donors are prevented by anti- HBc screening. Anti-HBc-positive donors unequivocally positive for anti- HBs should be considered noninfectious for HBV and should be allowed to donate. Anti-HBc screening of paid plasmapheresis donors, supplemented by anti-HBs testing, would reduce the amount of HBV to be processed by virus inactivation and increase the content of anti-HBs in plasma pools.     

Mapping alveolar binding sites in vivo using phage peptide libraries

Targeting lung tissue is nonselective due in part to the lack of specific cell-surface receptors identified on target lung cells. We used in vivo phage display to identify a panel of peptides that can bind selectively to lung epithelial cells with less binding to nonepithelial cells. By direct intratracheal instillation of phage libraries into the lung, we isolated and identified 143 individual phage clones. Three phage clones revealed enhanced binding to the lung in vitro and in vivo. These three identified peptides were synthesized and demonstrated selective binding to epithelial cells in lung tissue versus the control peptide. Further, the peptides specifically bound to freshly isolated type II alveolar epithelial cells compared with Hep2 cells. The results suggest that the airway phage display approach could be exploited for analyzing the molecular diversity in the lower respiratory tract.     

Identification of a motif for HLA-DR1 binding peptides using M13 display libraries.

Oligonucleotides encoding peptides known to bind to HLA-DR1 molecules have been inserted into the gene III of filamentous M13 phages. DR1 molecules purified from human lymphoblastoid cell lines could specifically bind to these peptide sequences expressed on the phage surface. A M13 phage peptide library was next constructed and screened with DR1 molecules. After four rounds of selection, more than 80% of the phages were able to bind to DR1. Competition experiments with both isolated phages and corresponding synthetic peptides showed that the binding was specific. Sequence analysis of the peptide encoding region of 60 phages binding to DR1 molecules and comparison with phages of the original library revealed two potential anchor positions. The first was an aromatic residue (Tyr, Phe, or Trp) at the NH2 terminus of the peptide sequences, and the second was located three residues downstream and consisted of Met or Leu. In addition, the negatively charged amino acids Asp and Glu were mostly excluded from the DR1 binding sequences, and the small amino acid residues Gly and Ala were enriched at position 6. As for DR1, this approach should enable one to easily determine the binding motifs of other MHC class II alleles and isotypes. Furthermore, it could have interesting applications in the design of major histocompatibility complex-specific antagonists.