| 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. |
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