【作者】 李文涵；

【导师】 沈关心；

【作者基本信息】 华中科技大学 ， 免疫学， 2007， 博士

【摘要】 肝癌的发生、发展过程非常复杂,涉及到肝癌细胞多分子的调变,利用不同的新技术和新方法或者将其排列组合,对肝癌细胞或肝癌组织进行鉴定筛选,在肝癌诊疗研究中具有重要的意义。表面展示技术(surface display)在筛选新配体方面显示了潜在的应用价值。本课题应用FliTrxTM细菌表面展示的随机十二肽库(FliTrxTM random peptide library),通过活细胞筛选获得能与人肝细胞肝癌细胞系HepG2特异性结合的多肽,为肝癌的靶向诊断和靶向治疗研究奠定实验基础。【目的】从FliTrxTM细菌表面展示的随机十二肽库中筛选与人肝癌细胞系HepG2特异性结合的十二肽,鉴定其结合特性,利用生物信息学工具分析其结构,为寻找肝癌细胞表面特异性标志以及肝癌的靶向诊断与靶向治疗研究奠定基础。【方法】1.肽库的筛选以正常肝细胞系L02为减差筛选的对照细胞,肝癌细胞系HepG2为筛选的靶细胞,从FliTrxTM细菌表面展示的随机十二肽库中筛选与HepG2特异性结合的短肽。经过五轮减差筛选,从最后一轮洗脱重收获的细菌中随机挑选700个单克隆细菌,通过菌落PCR筛选其中含有完整融合基因的克隆。2.短肽的特异性鉴定与序列分析以L02细胞为对照,通过流式细胞术(FCM)检测单克隆和HepG2的结合特异性;阳性克隆的测序结果与蛋白数据库进行比对和同源性分析。进一步采用FCM鉴定单克隆十二肽与不同恶性肿瘤细胞系的结合特性,包括胃癌细胞系MKN28、乳腺癌细胞系MCF7、宫颈鳞状细胞癌细胞系Hela、早幼粒急性白血病细胞系HL60、慢性骨髓性白血病细胞系K562、肺鳞状上皮细胞癌SLC等。3.结构预测利用Swissprot蛋白数据库的ProtParam工具,初步分析特异性多肽的氨基酸序列及稳定性;利用EMBL蛋白数据库的AGADIR工具,预测其可能的二级结构;利用Swissprot蛋白数据库的3Djigsaw蛋白质三级结构预测服务器模建短肽-TrxA融合蛋白的空间构象。4.结合的靶分子分析利用SDS-PAGE优化诱导细菌表达多肽的条件,进一步在Western-Blot中用可溶性短肽-TrxA融合蛋白检测多肽与肝癌细胞膜蛋白的结合,以L02和SLC细胞为对照,分析多肽特异性识别的肝癌细胞表面分子。【结果】1.肽库的筛选以正常肝细胞系L02和肝癌细胞系HepG2为筛选靶细胞,对FliTrxTM细菌肽库进行五轮差异筛选,从最后一轮筛选得到的多克隆中随机挑选700个阳性单克隆,菌落PCR结果表明,其中200个克隆含有完整的融合蛋白基因。2.短肽与肝癌细胞系结合的特异性FCM证实200个克隆中10个克隆表达的短肽可与肝癌细胞系HepG2相对特异性结合;经DNA测序,基因序列分析,确定7个阳性克隆,分别插入不同序列的随机肽片段。7个短肽的基因序列中存在出现频率较高的位点,但与Pubmed,Swissprot和EMBL的蛋白数据库进行比对和同源性分析,并未发现具有高度同源性的序列。FCM检测7个短肽与不同恶性肿瘤细胞系(包括胃癌、乳腺癌、宫颈鳞状细胞癌、早幼粒急性白血病细胞系、慢性骨髓性白血病细胞系、肺鳞状上皮细胞癌)结合的特性,发现其中三个短肽只显示出与肝癌细胞系相对较高的结合能力,命名为Hep1,Hep2和Hep3。3.结构分析预测结果利用ProtParam工具分析发现,Hep1和Hep2的结构相对不稳定,而Hep3具有相对稳定的线性结构。AGADIR工具对二级结构的预测提示,Hep1和Hep2可能形成α螺旋,而Hep3不会形成α螺旋。3Djigsaw同源模建得到的三级结构提示短肽-TrxA融合蛋白中的短肽均形成突出于融合蛋白之外的空间构象,提示由TrxA形成的随机肽的环状结构在与靶分子的识别中起到了重要的作用。4.十二肽结合的靶分子利用可溶性Hep1,Hep2和Hep3与TrxA的融合蛋白检测肝癌细胞,以SLC和L02细胞作为对照细胞,随机挑取的无关单克隆短肽-TrxA融合蛋白作为对照检测分子,Western-Blot结果显示Hep1,Hep2和Hep3三种不同的短肽-TrxA融合蛋白均可以识别HepG2细胞膜蛋白中大小约为140kDa的分子片段,而与SLC和L02反应未见特异性条带。随机可溶性短肽-TrxA融合蛋白与肝癌细胞、SLC和L02细胞反应均为阴性。【结论】以人肝细胞系L02为对照,人肝癌细胞系HepG2为靶细胞,通过五轮减差筛选,从细菌鞭毛展示的随机肽库中获得与HepG2特异性结合的十二肽,检测短肽-TrxA融合蛋白与肝癌细胞结合的特性。结果表明,获得3个FliTrxTM细菌表面展示的随机十二肽具有与HepG2细胞特异性结合的能力,而与其他肿瘤细胞和正常肝细胞呈阴性反应。Western-Blot检测结果表明,Hep1,Hep2和Hep3三种不同的可溶性短肽-TrxA融合蛋白均可与肝癌细胞表面靶分子结合,其分子量约为140kDa,可能为肝癌细胞特异性表达分子。更多还原

【Abstract】 Hepatocarcinogenesis is a slow multistep and multifactorial process, usually a consequence of long-term inflammation and fibrosis, which involves the progressive accumulation of changes at the level of gene and protein expression. The identification of novel tumor biomarker is pivotal for progression in the fields of HCC immunotherapy and diagnosis. Surface display techniques have successfully been applied to identify target-specific molecules such as antibodies or peptides. The principle advantage of display library methodologies is that selection can be performed in native-like, membrane-bound environment without a priori knowledge of the target cell receptors. Antibody and peptide ligands generated in this manner have been proven useful for in vivo imaging studies, therapeutic targeting and for identification of cell specific surface markers.As target molecules, peptides show advantages due to their smaller size than the other molecules, such as antibodies. Localization of peptides is not limited by diffusion, and clearance from the circulation is rapid, resulting in low background activity. Furthermore, the binding affinities of peptides are similar to those observed with antibodies. High affine receptors for peptides have been reported in a variety of tumors and can be used as molecule targets for image diagnosis and treatment of tumors.The bacterial FliTrx system (Invitrogen) was used in the present study to identify peptides specific to human hepatocellular carcinoma cell HepG2. In this flagella display library, peptides are directly displayed on the surface of E. coli fused with 2 proteins: the major bacterial flagellar protein (FliC) and thioredoxin (TrxA). Random dodecapeptides are cloned into the frame within the active loop of TrxA, which forms a stable protruding from the bacterial cell surface with the help of bacteria flagella.Objective: To identify peptides specific for hepatoma cell line HepG2 by biopanning from the bacterial FliTrx system, to find the potential HCC associated target molecules for HCC diagnosis and therapy.Methods: FliTrx bacterial display library was used to search for HCC specific peptides by biopanning with the hepatocellular carcinoma cell line HepG2 using the healthy liver cell line L02 as the counter-selecting control. After five rounds of biopanning,700 individual clones were picked up. Bacterial polymerase chain reaction (PCR) was carried out to identify the clones containing dodecamer fusion sequence. The peptides specific for HepG2 were selected by flow cytometry (FCM). Positive clones were sequenced using Applied Biosystem Automated DNA sequencers 3730. Sequences of dodecapeptides were blast in database of Pubmed, Swissprot and European Molecular Biology Laboratory. Primary structure of the peptides was analized with ProtParam tool and the secondary structure prediction was performed with AGISR tool on the internet. The whole three-dimensional structure of peptide-TrxA fusion proteins was modeled with tertiary analysis tool 3Djigwaw tool. Furthermore we used the soluble peptide-TrxA fusion protein to identify the potential target molecules on hepatocellular carcinoma in Western-Blot.Results: Three different positive clones were obtained, named Hep1, Hep2 and Hep3. These sequences are not similar to hepatocellular specific antigen or any other peptide or protein sequences available, as confirmed by blast in various protein databases.Computer graphic modeling pointed out that the structure of peptide-TrxA fusion proteins was important for the function of peptides. Results of western-Blot indicated the protein, which soluble peptide-TrxA fusion proteins recognized, was about 140kDa.Conclusions: Bacterial peptide library is a novel approach to isolate specific peptides binding direct to tumor cells, even to identify the target molecule on tumor cells. The bacterial display random peptide library biopanning on living cells permits identification of specific peptides for HepG2 cells.更多还原