噬菌体随机12肽库筛选肝癌患者血清肿瘤标记物的初步研究

目的:从噬菌体随机多肽文库中,筛选出能与肝癌患者血清特异性结合的短肽分子.方法:采用肝癌患者血清作为配基,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筛过程,经3轮淘筛后,随机挑取噬菌体克隆用ELISA检测其特异性,评价分析其诊断肝癌的价值.结果:经3轮淘筛后,特异性结合的噬菌体富集增加近100倍.用.ELISA检测第3轮筛选后随机挑取的单个噬菌体克隆,其中特异性最好的3个克隆具有诊断肝癌的潜在价值.结论:噬菌体展示肽库技术,可以有效进行肝癌相关抗原肽的筛选研究,为获得特异性诊断试剂进而为肝癌的诊断提供依据.     

MDA-MB-231乳腺癌细胞特异性多肽的初步筛选

目的 利用噬菌体展示技术,从噬菌体随机十二肽库中筛选出能够特异性结合MDA-MB-231乳腺癌细胞的噬菌体克隆.方法 以人正常乳腺细胞为减性筛选细胞、MDA-MB-231乳腺癌细胞为靶细胞,对噬菌体随机十二肽库进行筛选,挑取富集后的阳性单克隆噬菌体,酶联免疫吸附试验（ELISA）及DAB染色鉴定阳性噬菌体的特异性及亲和力.结果 经过3轮筛选,噬菌体得到约113倍的富集,随机挑选11株单克隆噬菌体,ELISA显示8号噬菌体单克隆对乳腺癌细胞的亲和力是对照的6.5倍,DAB鉴定亦显示其对乳腺癌细胞的特异性及亲和力最高,命名为LK-8.结论 利用噬菌体筛选技术成功筛选出能够特异性结合MDA-MB-231乳腺癌细胞的特异性噬菌体单克隆LK-8,可为进一步合成特异性多肽用于早期诊断和靶向治疗乳腺癌奠定基础.     

噬菌体肽库在宫颈癌血清肿瘤标志物筛选中的应用

目的:用噬菌体随机12肽库对宫颈癌患者血清进行差异性筛选,筛选出能与宫颈癌患者血清特异性结合的肿瘤标志物。方法:应用噬菌体随机12肽库对宫颈癌患者和正常人血清进行三轮差异性筛选,用ELISA法检测噬菌体克隆对宫颈癌患者血清结合的特异性。结果:经过三轮筛选,噬菌体富集率逐轮提高,提高近100倍。用ELISA法对50例宫颈癌患者血清和50例健康者血清检测验证,其中获得一株与宫颈癌患者血清特异结合较好的噬菌体,对宫颈癌的早期诊断具有潜在价值。结论:筛选出能与早期宫颈癌患者血清高亲和力特异结合的短肽,为研究宫颈癌肿瘤标志物及宫颈癌的早期诊断奠定了基础。     

利用体内噬菌体展示技术筛选肝癌组织特异性结合肽

目的 筛选人源肝癌组织特异性结合短肽。方法 体外培养人源肝癌细胞株BEL-7402,建立荷瘤裸鼠模型。尾静脉注射噬菌体12肽文库至荷瘤裸鼠体内,循环20min后回收肿瘤组织中噬菌体,同时取正常对照组织进行噬菌体效价测定和免疫绀化观察。将同收的噬菌体扩增、纯化,并以此作为起始物进行下一轮筛选。经过3轮体内筛选,得到与肝癌组织或细胞特异结合的肽段。随机挑选噬菌体单克隆进行测序,分析序列㈣源性后进行体外细胞酶联免嫂吸附试验（ELISA）和体内回输实验,验证噬菌体克隆的导向性。结果 经过3轮体内筛选,瘤组织中噬菌体的回收率逐步提高,回收量随着输入量的增加迅速增加,而每轮筛选肝组织中的噬菌体回收晕始终保持在一个恒定范围,并不随输入量的增加而增加。免疫组化结果硅示,第3轮筛选后,瘤组织中的噬菌体得到高水平富集,同时其他组织的非特异性结合降至最低,肝癌细胞特异性结合最强的是A54号单克降,A67、B2号单克隆次之。B2的导向效果最好,A54次之。通过对噬菌体单克隆的序列分析,初步确定了PSS／FTT基序。结论 利用体内噬菌体展示技术,可以成功筛选到与肝癌细胞或组织特异结合的噬菌体肽。     

宫颈癌HeLa细胞特异性结合肽的筛选及鉴定

背景与目的:宫颈癌的分子靶向治疗具有很好的疗效,同时可以显著减少抗癌药物对人体自身的损伤,因此备受关注。本研究利用噬菌体体内展示技术筛选及鉴定宫颈癌特异性结合肽,将有可能成为化疗药物的靶向载体,为宫颈癌靶向药物治疗奠定基础。方法:体外培养宫颈癌HeLa细胞接种裸鼠,建立肿瘤动物模型。将随机肽库尾静脉注入裸鼠体内,循环15 min,心脏灌注后回收肿瘤组织噬菌体扩增、纯化并以此作为起始物进行第2轮的筛选,如此进行3轮体内筛选后挑取噬菌体克隆,进行免疫组化及ELISA实验,初步鉴定噬菌体克隆对宫颈癌细胞的亲和力及特异性,并将具有强亲和力的克隆进行测序。结果:ELISA结果显示,随机挑选10个噬菌体单克隆中8个克隆对HeLa细胞具有很强的亲和力,将这8个克隆进行测序,获得相同短肽序列LLRSTGF。结论:利用噬菌体展示技术筛选出与宫颈癌细胞HeLa特异性结合的短肽,进一步与化疗药物结合,为宫颈癌靶向治疗提供新的方法。     

随机十二肽噬菌体展示文库筛选血型B抗原模拟多肽的实验研究

目的:筛选出替代血型B抗原的模拟多肽,用多肽抗原替代糖类抗原.方法:抗血型B抗原的单克隆抗体作为固相筛选靶分子,对随机十二肽噬菌体展示文库进行生物淘选(bio-panning),经包被-结合-洗脱-扩增等循环3轮,对筛选的克隆ELISA鉴定,并通过剂量依赖实验验证其结合特异性.最后提取DNA测序,确定模拟肽氨基酸序列.结果:3轮筛选结束,得到2个亲和力较强的十二肽序列TKNMLSL-PVGPG和HSLKHTQMSYSS.结论:经过生物筛选得到模拟多肽序列,利用噬菌体展示技术筛选糖类抗原的模拟肽具有可行性,为糖类抗原的研究提供一种新思路.     

胃癌SGC7901细胞特异性结合肽的筛选及鉴定

目的筛选与人胃癌细胞株SGC7901特异性亲和的短肽,提高胃癌治疗的针对性。方法建立裸鼠胃癌模型,将噬菌体随机环7肽文库通过尾静脉注射入裸鼠体内,每轮筛选后测噬菌体效价。采用免疫组化及ELISA等方法,对目标噬菌体进行检测,提取噬菌体DNA,进行序列测定。结果经过4轮筛选,噬菌体在胃癌肿瘤组织中得以明显富集。免疫组化结果显示肿瘤组织为强阳性。ELISA结果显示:随机挑选的13个克隆中12个对SGC7901细胞的亲和力高。序列测定结果:PPRWMPS出现的次数最多。结论提示短肽PPRWMPS可能对荷瘤裸鼠模型的胃癌组织具有很强地结合力,有望成为胃癌治疗的靶点。     

应用噬菌体随机肽库筛选肿瘤MUC1/Y黏蛋白结合肽

目的：探索用噬菌体随机肽库筛选可用于肿瘤导向治疗的新型小分子载体。方法：以MUC1/Y黏蛋白的胞外段蛋白（MUC1/Yex）为靶分子,用凝胶亲和法和酶联板法分别筛选十二肽噬菌体随机肽库,ELISA鉴定阳性克隆,DNA序列测定后确定MUC1/Yex结合肽的氢基酸序列;免疫组化鉴定阳性噬菌体克隆及正常及肿瘤细胞的结合能力及特异 性。结果：通过4轮筛选,共获得3种MUC1/Yex的结合肽,分别为HHWHSRSQLSWF,HLKHKNYLPPTP和GNWYRHPHYLQP,其中HXXHS表位可能在与MU1/Yex的结合中起重要作用。阳性噬菌体克隆可与肿瘤细胞系MCF7,OVCA3,而不与正常外周血淋巴细胞结合。结论：筛选获得的MUC1/Yex结合肽具有一定的亲合力和肿瘤特异性,可进一步用于肿瘤导向治疗的研究。     

抗内毒素噬菌体抗体库的构建及筛选

目的构建一个鼠源性的抗内毒素单链噬菌体抗体库,从中筛选出对内毒素具有较高亲和力的单链抗体。方法从小鼠脾细胞中提取总RNA,通过RT-PCR技术扩增出小鼠抗体重链、轻链可变区基因（VH,VL）,用Linker将VH,VL交联形成单链抗体可变区片段（ScFv）。经NotⅠ,SfiⅠ双酶切后与经同样双酶切的pCANTAB5E载体相连,转化入大肠杆菌TG1以构建鼠抗内毒素单链噬菌体抗体库。在援救噬菌体抗体库后,用内毒素淘筛特异性的ScFv,富集的噬菌体阳性克隆重新感染TG1。在96孔板分别援救单个含特异性ScFv的TG1菌落,最后随机挑选出190个菌落经ELISA检测抗内毒素ScFv。结果小鼠血清中抗内毒素的效价为1∶12800。提取的总RNA浓度为12.3813μg/ml,纯度较好。扩增出的VH长约340bp,VL约320bp,ScFv约800bp。转化入TG1后有约1.9×107个菌落。淘筛一轮过后即有3×104阳性菌落长出,190个菌落经ELISA检测有2个阳性克隆。结论成功地构建了一个库容量为1.9×107的鼠抗内毒素单链噬菌体抗体库,并从中筛选出了2株抗内毒素ScFv。     

胃癌细胞特异性结合肽的筛选及鉴定

目的 筛选与胃癌细胞特异性结合的多肽。方法 以正常细胞为吸附细胞,胃癌细胞为筛选靶细胞对噬菌体随机12肽库进行消减筛,用细胞酶联免疫吸附试验（ELISA）、免疫细胞和组织化学法及裸鼠正常组织结合实验鉴定阳性克隆并进行DNA测序。结果 经三轮筛选,利用ELISA从随机挑选的24个噬菌体克隆中得到8个与胃癌细胞具有高结合力的噬菌体阳性克隆,经免疫细胞化学及裸鼠正常组织结合试验鉴定,发现第20、24两个克隆能与胃癌细胞特异性结合,而不与正常细胞和裸鼠组织结合,噬菌体阳性克隆氨基酸序列无同源性。结论 得到两个序列不同的特异性结合胃癌细胞的噬菌体克隆,这可为进一步的研究提供实验依据。     

In Vivo Selection of Phage Sequences and Characterization of Peptide-specific Binding to Breast Cancer Cells

OBJECTIVE To screen specific polypeptide target binding to breast cancer xenogra s in vivo from a phage-displayed peptide library in order to provide peptide sequences for breast cancer tumor-targeting diagnosis and therapy. METHODS A mouse model for carrying breast cancer xenografts was established using Tientsin Albinao II mice(TA II).A 12-peptide library was biopanned through 4 rounds. Phages were recovered and titrated from tumor xenografts and control tissue(liver).The distribution of phages was detected by immunohistochemical staining. RESULTS Phage homing to breast cancer was enriched through 4 rounds of biopanning,being 14-fold of that recovered from liver tissue.A peptide sequence,ASANPFPTKALL was characterized by randomly picked-up clones which appeared most frequently. Immunohistochemical staining revealed phage localization in cancer xenografts 40 min after injection of the enriched phages. When a specific phage was tested individually,the phage reclaimed from breast cancer xenografts was 14 times as those from control tissues. CONCLUSION Tumor-specific homing peptides may provide an effective tool for breast cancer target therapy.The in vivo phage display selection technique employed in this study was feasible and applicable to screening peptides that home to breast cells.     

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency oftraditional techniques both in diagnosis and therapy of the disease makes the development of alternative and noveltechniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be usedto increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of thisstudy is screening and identification of individual peptides specifically targeted to human gastric cancer cells usinga phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptidesequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cellswere used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide librarywere applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were establishedby enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clonesafter five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequenciesof each amino acid in best binding clones to determine positively overexpressed amino acids for designing novelpeptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific bindingon MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acidfrequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, butmore importantly, data extracted from eluted phage clones may be used to design theoretical peptides with betterbinding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potentialcandidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research.     

Screening peptides binding specifically to colorectal cancer cells from a phage random peptide library

The aim of this study was to screen for polypeptides binding specifically to LoVo human colorectal cancer cells using a phage-displayed peptide library as a targeting vector for colorectal cancer therapy. Human normal colorectal mucous epithelial cells were applied as absorber cells for subtraction biopanning with a c7c phage display peptide library. Positive phage clones were identified by enzyme-linked immunosorbent assay and immunofluorescence detection; amino acid sequences were deduced by DNA sequencing. After 3 rounds of screening, 5 of 20 phage clones screened positive, showing specific binding to LoVo cells and a conserved RPM motif. Specific peptides against colorectal cancer cells could be obtained from a phage display peptide library and may be used as potential vectors for targeting therapy for colorectal cancer.     

环7肽库筛选胃癌耐药细胞特异性结合短肽

目的获得与胃癌耐药细胞株SGC7901/VCR特异结合的抑制耐药短肽,作为提高胃癌化疗效果的先导化合物。方法以SGC7901/VCR细胞为靶细胞,胃粘膜永生化上皮细胞GES,胃癌药敏细胞SGC7901为吸附细胞对噬菌体7肽库进行消减筛选,用细胞ELISA鉴定阳性噬菌体克隆并测序,利用竞争抑制试验确定阳性克隆的结合部位是否为外源性肽段。结果经4轮筛选,从随机挑选的30个噬菌体克隆中得到14个能特异性与SGC7901/VCR结合而不与胃癌药敏细胞结合的阳性克隆,确定其氨基酸序列分别为SY1和SY2(筛选所得安基酸序列正在专利申请中),含SY1为阳性克隆。结论用噬菌体环7肽库成功筛选到能特异性结合胃癌耐药细胞株的短肽,为肿瘤治疗或药物靶向治疗奠定基础。     

乳腺癌干细胞富集及其特异性多肽的筛选

目的：无血清培养法富集乳腺癌干细胞（breast cancer stem cell,BCSC）并采用噬菌体展示技术,筛选能特异性结合乳腺癌干细胞的噬菌体多肽。方法：无血清培养法富集乳腺癌MDA—MB-231细胞株中干细胞并以此为靶标,以hs578bst人正常乳腺细胞及普通培养的MDA—MB-231细胞为减性筛选细胞,对噬菌体随机肽库进行双重减性筛选,选取富集后的阳性噬菌体单克隆,ELISA及DAB染色鉴定阳性噬菌体特异性并测序。结果：经过3轮筛选,噬菌体得到约500倍的富集,随机挑选10株单克隆噬菌体。ELISA显示,6号噬菌体单克隆对乳腺癌干细胞的亲和力是对照的6．14倍;DAB鉴定亦显示,其对乳腺癌干细胞的特异性及亲和力最高,对阳性噬菌体DNA测序翻译得到十二肽氨基酸为GYSASRsTIPGK。结论：通过干细胞富集及噬菌体展示技术,成功筛选出能够特异性结合乳腺癌干细胞的特异性噬菌体多肽,为乳腺癌的干细胞靶向治疗和深入研究奠定基础。     

Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library

Objective

Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy.

Methods

The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides.

Results

After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with K_aff (affinity constant) of 16.4±8.6 µg/mL (n=3), 9.2±2.1 µg/mL (n=3), and 174.8±31.1 µg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed.

Conclusion

These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.     

Isolation, characterization, and recovery of small peptide phage display epitopes selected against viable malignant glioma cells

Phage display techniques rely on nearly random oligonucleotide sequences inserted into the protein III filament binding protein of an Escherichia coli filamentous phage M13 to generate a library of phage that express more than 10(7) different peptides. Phage that expresses a sequence having high affinity for a specific molecule, cell, or tissue can then be isolated through selective binding and recovery. Selected phage cannot only be used as gene transfer vectors in themselves, but the small peptide epitopes can be sequenced and potentially recombined into the attachment proteins of viral vectors, or used by themselves to target other therapeutic agents and diagnostic imaging radiolabels. Most phage display selections are carried out against purified and/or fixed protein targets, raising concerns as to the relevance of the selected epitopes. We have selected phage from the CMTI library against viable U87-MG human malignant glioma cells using a derivation of biopanning. The library, which initially contained phage expressing 2x10(7) different epitope sequences, collapsed after four rounds of selection such that 42% of recovered clones expressed a consensus sequence. Selective binding to viable adherent U87-MG cells was subsequently demonstrated under physiologic conditions at 167% (+/-27%) unselected phage using a novel, viable enzyme-linked immunosorbent assay technique. In comparison, there was no difference in binding to control 9L rat gliosarcoma, PANC-1 human pancreatic adenocarcinoma, T98-MG human malignant glioma, or AST-4 human malignant glioma cells of selected compared to unselected phage. Using polymerase chain reaction, the epitope was recovered with flanking unique restriction sites for recombination into a herpes simplex virus type-1 vector. This study demonstrates and discusses optimized methodologies for using phage display to target viable cells.