Keratinocyte growth factor (KGF) phage model peptides can promote epidermal cell proliferation without tumorigenic effect

Background KGF significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. ELISA was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. MTT assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results 56.9% (33/58) of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or EGF. MTT assay data showed that four (No.1-4) of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor (KGFR) mRNA in the KGF control group and the two phage model peptide groups (No.1 and No.2) increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups (No.1-4). Conclusions Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.     

Transforming growth factor-β1 phage model peptides isolated from a phage display 7-mer peptide library can inhibit the activity of keloid fibroblasts

Background Transforming growth factor-β1 (TGF-β1) is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB (NF-κB) mRNA, connective tissue growth factor (CTGF) mRNA and TGF-β receptor Ⅱ (TβRII) mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide (No. 4) could promote keloid fibroblasts proliferation,however, three phage model peptides (No. 1-3) could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.     

转化生长因子β1噬菌体模拟肽抑制瘢痕疙瘩成纤维细胞增殖的实验研究

目的 从噬菌体随机12肽库中筛选获得转化生长因子(TGF)β1噬菌体模拟肽,评估其抑制瘢痕疙瘩成纤维细胞增殖的作用.方法 以人TGF-β1单克隆抗体为靶,生物淘选噬菌体模拟肽.噻唑蓝(MTT)比色法定量测定活细胞的数量.Annexin V-FITC/PI凋亡检测试剂盒和流式细胞仪检测对成纤维细胞的凋亡作用.免疫荧光测定检测模拟肽与成纤维细胞的亲和力.实时定量PCR分析方法检测成纤维细胞核因子κB(NF-κB),结缔组织生长因子(CTGF)的表达水平.结果 共获得10种噬菌体模拟肽,具有与TGF-β1、TGF-β2、TGF-β受体Ⅱ(TβRⅡ)、TGF-β诱导因子、NF-κB或细胞分裂原素活化蛋白激酶(MAPK)相似的序列.MTT比色测定结果显示4种(第7～10组)噬菌体模拟肽组能够抑制瘢痕疙瘩成纤维细胞增殖(P＜0.05).免疫荧光测定显示是噬菌体上的模拟肽,而不是噬菌体本身,能够与瘢痕疙瘩成纤维细胞相结合;凋亡实验显示这4种噬菌体模拟肽能够轻度促使瘢痕疙瘩成纤维细胞发生轻度的晚期凋亡;实时定量PCR的结果显示这4种噬菌体模拟肽NF-κB的表达降低,表达量分别是阴性对照组的0.28、0.26、0.46、0.30倍,4种噬菌体模拟肽CTGF的表达降低,表达量分别是阴性对照组的0.26、0.60、0.34、0.17倍.结论 噬菌体模拟肽可能是通过调节NF-κB及CTGF的表达,调节瘢痕疙瘩成纤维细胞的增殖.     

应用噬菌体随机十二肽库筛选hFGF 7相关模拟肽

目的应用噬菌体随机十二肽库生物淘选具有刺激表皮细胞增殖作用的人成纤维细胞生长因子 7(hFGF 7)噬菌体模拟肽。方法以hFGF 7单克隆抗体为靶,进行4轮生物淘选噬菌体随机十二肽库;随机挑取单克隆噬菌体,应用ELISA方法检测单克隆噬菌体的结合力,对结合力较好的单克隆噬菌体提取噬菌体DNA,进行测序,并行序列分析。结果经过4轮生物淘选,特异性噬菌体模拟肽获得了富集,ELISA检测结果显示获得的一些噬菌体模拟肽具有较好的结合力,测序获得11个与促进细胞分裂、增殖或抑制细胞增殖有关的碱基序列。结论从噬菌体随机十二肽库中可淘选到与hFGF 7相关的噬菌体模拟肽,其中部分噬菌体模拟肽可能会促进表皮细胞增殖,期望将来可应用于促进创面愈合。     

噬菌体肽库筛选血小板衍生生长因子受体β链(PDGFRβ)亲和短肽及其抗肝纤维化实验研究

目的:从噬菌体随机肽库中筛选血小板衍生生长因子受体β链(PDGF-Rβ)的亲和短肽并探讨其对CC14诱导的肝纤维化模型中的抗肝纤维化作用.方法:以重组可溶性人PDGF-Rβ作为靶标,应用噬菌体随机十二肽库进行筛选,经过3轮淘选,提取阳性噬菌体克隆ssDNA,测序并进行序列分析,选择其中出现频率高的展示肽,并根据其氨基酸...     

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 de-velope 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 ELISA 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.     

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.     

Identification and Characterization of Peptide Mimics of Blood Group A Antigen

In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase （GST） fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.     

噬菌体展示技术筛选卵巢癌细胞表面转移相关分子结合肽

目的：利用噬茵体7肽库筛选高转移潜能卵巢癌细胞株HO-8910PM表面转移相关分子结合肽。方法：利用噬茵体展示技术体外快速差减筛选（biopanning and rapid analysis of selective inter-activeligands,BRASIL）卵巢癌细胞株HO-8910和HO-8910PM,经过连续5轮生物淘洗,随机挑选14个噬茵体单克隆进行DNA测序,并进行ELISA、噬茵体竞争结合实验验证阳性噬茵体的亲和性。结果：噬菌体克隆Z3（短肽LRLRNTR）对高转移潜能卵巢癌细胞株HO-8910PM有较高的亲和性。结论：噬茵体展示技术筛选的短肽LRLRNTR可望为卵巢肿瘤早期诊断、转移复发及治疗提供新的方向。     

细胞间黏附因子1(Ⅰ区)模拟肽的筛选及初步鉴定

目的 筛选表达细胞间黏附因子1（ICAM－1）（Ⅰ区）模拟肽的特异噬菌体克隆,为研制抗脑型疟黏附肽类药物奠定基础。方法 以抗ICAM－1（Ⅰ区）的单抗15．2为靶,采用亲和筛选法对噬菌体随机十二肽库进行3轮筛选,通过ELISA及竞争抑制试验初步鉴定了获得的噬菌体短肽与单抗15．2之间的结合特性。结果 从第3轮洗脱液中挑选20个噬菌体克隆进行夹心法ELISA检测,其中18个克隆OD值超过阴性对照2．1倍以上,阳性率达90％。竞争性ELISA试验表明多数阳性噬菌体与ICAM－1能竞争性地与15．2单抗结合。结论 阳性噬菌体表达的短肽可能是15．2单抗所识别的模拟表位。Ⅰ     

从噬菌体随机十二肽库中筛选乙酰胆碱酯酶的抑制肽

目的从随机肽库中筛选乙酰胆碱酯酶（ACHE）的抑制性多肽。方法以人AChE作为靶标，应用噬菌体随机12肽库进行筛选，经过3轮筛选，对阳性克隆进行测序并进行序列分析及抑酶活性测定。结果筛选得到6个能与人AChE较强结合的噬菌体克隆，其中有4个克隆可以抑制AChE的酶活性，其保守序列为W（S／P）HY。结论通过噬菌体肽库技术能够筛选到抑制人AChE活性的多肽，为进一步研究AChE的多肽抑制剂奠定了基础。     

从噬菌体展示肽库中筛选 PreS1抗原的结合肽

目的:从噬菌体随机七肽库中筛选能与PreS1抗原特异性结合的多肽。方法:利用噬菌体展示技术,以PreS1抗原为靶分子,从随机七肽库中进行生物亲和筛选,ELISA鉴定阳性克隆。结果:对肽库进行3 轮筛选后,通过ELISA和竞争抑制ELISA,获得了特异性的结合肽,测序结果显示噬菌体上的短肽有共同序列。结论:利用噬菌体展示技术成功筛选到了PreS1抗原的结合肽,为乙肝的治疗和诊断探索新的途径。     

噬菌体表面展示文库筛选人干细胞因子模拟肽

目的 用噬菌体表面展示文库筛选具有人干细胞因子(hSCF)生物学活性的模拟肽.方法 采用酶联免疫吸附测定法(EUSA)从噬菌体环七肽库和十二肽库筛选与重组hSCF受体(rc-kit/Ig1-3)有较高亲和力的噬菌体克隆,提取噬菌体单链DNA进行序列测定,根据序列测定结果合成阳性小肽,四甲基偶氮噻唑盐(MTT)法检测合成小肽刺激UT-7细胞增殖的活性.结果 经3轮筛选获得11个来源于环七肽库和8个来源于十二肽库与rc-kit/Ig1-3结合活性较高的噬菌体克隆,DNA测序结果显示环七肽库筛选到1个共有序列DPSSPHTH,十二肽库没有筛选到共有序列.序列比对分析显示,这些小肽与hSCF没有同源序列.MTT法测定结果表明,合成的4个小肽均能促进UT-7细胞增殖,其中CE16和LE20刺激效果明显.结论 获得4个具有较高hSCF生物学活性的模拟肽.     

能结合HCV E2蛋白的人CD81模拟肽的筛选与活性鉴定

目的用CD81单克隆抗体筛选噬菌体随机展示十二肽库,以期得到可模拟人CD81功能位点、能抑制HCVE2与其受体—人CD81分子结合的小肽。方法用CD81单抗从噬菌体随机展示十二肽库中筛选人CD81模拟肽,用ELISA鉴定阳性噬菌体克隆;以阳性噬菌体免疫BALB/c小鼠,分析小鼠免疫血清对阳性噬菌体与HCVE2结合的阻断作用;以HCVE2蛋白包被酶标板,检测阳性噬菌体对HCVE2与人CD81分子结合的抑制作用。结果对十二肽库进行3轮筛选后,经ELISA和DNA测序,鉴定出3个(C4,C13和C16)阳性克隆,与人CD81无同源序列。阳性噬菌体克隆C16免疫小鼠血清能阻断该克隆与HCVE2的结合。C16克隆能以剂量依赖的方式竞争性抑制HCVE2蛋白与人CD81的结合。结论用人CD81单抗从噬菌体随机展示肽库中筛选出的阳性噬菌体克隆所编码的小肽在功能上能模拟人CD81与HCVE2的结合活性,能竞争性抑制HCVE2与人CD81分子的结合,在抗HCV药物及疫苗研究中具有潜在应用价值。     

利用Sm抗原模拟表位肽构建狼疮样鼠模型

目的利用Sm抗原模拟表位肽免疫BALB/C小鼠,构建狼疮样鼠模型。方法用鼠Sm单克隆抗体对噬菌体随机12肽库进行亲和筛选,将筛选的噬菌体阳性克隆行双抗体夹心ELISA检测、DNA序列测定并推导其氨基酸序列,初步确定Sm抗原模拟表位;进而用混合噬菌体阳性克隆皮下免疫BALB/C小鼠,ELISA法检测小鼠血清IgG型抗Sm抗体、抗双链DNA（dsDNA）抗体和抗核抗体（ANA）水平;直接免疫荧光检测小鼠肾脏IgG型免疫复合物的沉积以及HE染色观察小鼠肾脏组织病理损伤情况。结果免疫筛选获得的5个ELISA强阳性的噬菌体克隆的氨基酸序列IR、SQ、PP出现频率增加;实验组小鼠血清中第28天出现Sm抗体、dsDNA抗体、ANA抗体,且滴度不断升高;第33天出现蛋白尿;肾脏可以检测到IgG型免疫复合物沉积及明显的肾脏病理改变。对照组小鼠无明显自身抗体产生和肾脏病变。结论利用Sm抗原噬菌体模拟表位肽可成功构建狼疮样小鼠模型。     

骨肉瘤细胞特异性结合短肽的筛选

目的获得与骨肉瘤细胞株os-732特异结合的短肽,作为骨肉瘤靶向治疗的先导化合物。方法以骨肉瘤细胞os-732为靶细胞,成骨细胞为吸附细胞对噬菌体12肽库进行差减筛选,用细胞ELISA、免疫组化鉴定阳性噬菌体克隆并测序。结果经三轮筛选,从随机挑选的20个噬菌体克隆中得到9个能特异性与骨肉瘤细胞os-732结合,而不与正常成骨细胞结合的阳性克隆。但其氨基酸序列无同源性。结论得到多个序列不同的特异性结合骨肉瘤的噬菌体克隆,提示骨肉瘤细胞表面结构复杂,具多个骨肉瘤抗原表位。本实验获得的短肽具有一定的亲合力和肿瘤特异性,为针对不同位点的靶向药物设计提供了实验依据。     

噬菌体肽库在筛选及鉴定高转移性肝癌特异性结合肽中的应用及其意义

目的:探讨噬菌体环七肽库在筛选与肝癌高转移细胞株HCCLM3特异高效结合的短肽中的应用,为进一步探索肝癌转移的分子机制和寻找肝癌转移标志物奠定基础。方法:以人肝癌高转移细胞株HCCLM3为靶细胞、人肝癌低转移细胞株SMMC7721为吸附细胞对噬菌体环七肽库进行4轮差减筛选,采用ELISA方法进一步筛选出与HCCLM3细胞高度亲和的阳性克隆(即实验组A450nm值高于对照组3倍以上),并利用免疫细胞化学方法鉴定噬菌体阳性克隆的特异性并测序。结果:经过4轮陶筛后,噬菌体在筛选靶细胞上出现明显富集,且逐轮提高(P<0.05);利用ELISA法对筛选后随机挑取的20个噬菌体克隆进行初步鉴定,得到6个能与肝癌细胞HCCLM3亲和度较高的阳性克隆(C4、C6、C7、C10、C11和C17);通过免疫细胞化学染色鉴定阳性克隆靶向肝癌细胞HCCLM3的特异性,与对照组比较,C7噬菌体对高转移潜能肿瘤细胞具有较高特异性(P<0.05);测序显示6个阳性克隆氨基酸序列无同源性。结论:利用噬菌体环七肽库筛选得到与人肝癌高转移细胞株HCCLM3具有较高亲和力的多肽。     

人骨肉瘤细胞特异性结合肽的筛选及验证

目的获得人骨肉瘤细胞MG63特异性结合肽,为骨肉瘤靶向治疗奠定基础。方法应用噬菌体展示技术,以人骨肉瘤细胞作为靶细胞,293T细胞为差减细胞,筛选获得MG-63细胞特异性结合肽。酶联免疫法进行靶向性验证。应用荧光染色技术初步探讨短肽细胞受体位置。制作人骨肉瘤模型,尾静脉注射目标噬菌体,免疫组化检测其靶向性。结果经过四轮一步有机相离心分离方法,获得了与骨肉瘤细胞特异性结合的短肽,并测序获得其中出现次数最多的序列SLTNLSK,靶向验证结果显示该短肽有较强的特异性。结论应用噬菌体展示技术,获得了与人骨肉瘤细胞特异性结合的短肽,序列为SLTNLSK,并验证了其靶向性。可以作为骨肉瘤靶向治疗的导向性化合物。     

人转铁蛋白受体结合肽的噬菌体肽库筛选与鉴定

目的：从噬菌体呈现12肽库中筛选与人转铁蛋白受体结合的肽．方法：以人转铁蛋白受体为靶蛋白，生物淘洗法从噬菌体呈现12肽库中筛选与之结合的阳性噬菌体，用噬菌体ELISA，FCM及免疫组化等方法检测阳性噬菌体与人转铁蛋白受体的结合活性．结果：从噬菌体肽库中筛选到-呈现SPRPRHTLRLSL肽的噬菌体，噬菌体ELISA，FCM及免疫组化等方法均证明呈现该肽的噬菌体可与人转铁蛋白受体或高表达人转铁蛋白受体的细胞结合．结论：SPRPRHTLRLSL具有与人转铁蛋白受体结合的活性，它为以转铁蛋白受体为靶点的导向性药物的开发提供了一个可能的线索．     

噬菌体肽库筛选隐丹参酮高亲和性结合肽

通过噬菌体十二肽库,以隐丹参酮为靶分子,筛选与隐丹参酮具有高亲和性的结合短肽。经过噬菌体十二肽库的3轮淘选,获得阳性噬菌体克隆,挑选结合力强的克隆进行测序,得到隐丹参酮的高亲和性结合短肽序列,并进行生物信息学分析。本试验共筛选到10个隐丹参酮的高亲和性结合短肽序列,有614种蛋白质与其结构相匹配。多肽序列的核心序列为VILDFGEI。本研究获得了与隐丹参酮结合的高亲和性多肽,为深入研究隐丹参酮的作用靶点以及分子作用机制提供了实验依据和结构基础。