Identification And Function Assay Of The Specific Peptide Aptamers Against The Interacting Domain Between CDP And SOX2 Protein

ObjectiveTo confirm the interacting domain between CDP and SOX2 protein, and to screen out specific peptide aptamers against the interacting domain between CDP-SOX2 protein. From a peptide aptamers library, which has a variable reading frame. Following this, to validate the bio-function of peptide aptamers after the establishment of KYSE450 stable cell lines, which can specifically express the target peptide aptamers. All our attempts will lay a foundation for the development of small molecule drugs against the esophageal squamous cell carcinoma(ESCC). Method1.We used high fidelity PCR and vector construction technology to obtained SOX2 and CDP full-length or truncated mutants fusion expression with Bimolecular fluorescence complementation(BiFc) vectors. Then we combined with BiFc analysis to visualize protein-protein interactions directly, and using co-immunoprecipitation technique to verify interacting domain between CDP and SOX2 protein.2.The peptide aptamers which against the CDP-SOX2 binding domain specifically were screened out from the aptamer library with BiFc, and the interaction between peptide aptamers and CDP-SOX2 bind domain were verified by the co-immunoprecipitation technique.3.We constructed the lentiviral vectors to express peptide aptamers, and established KYSE450-PEPTIDE APTAMER stable cell lines. The function of cell lines KYSE450-PEPTIDE APTAMER were preliminary explored by cell proliferation assay. Result1.Determination of the interacting domain: CDP and SOX2 full-length and truncated mutants recombinant plasmid which could be applied in BiFc were obtained by high fidelity PCR and vector construction technology, and got bands with expected length after recombinant plasmids were identified by restriction endonuclease digestion analysis; According to the results of BiFc we found there are interactions between CDP and SOX2 full length and truncated mutants; And using co-immunoprecipitation technique we verified the CDP-SOX2 binding domain locate in 1-100 amino acids of CDP protein.2.Screening of specific peptide aptamers : Recombinant plasmids pBiFcVN173-CDP?C(101-1516) and PCMV-Tag2B-PEPTIDE were obtained by high fidelity PCR and vector construction technology. After recombinant plasmids were identified by restriction endonuclease digestion analysis, we obtained the size of 300 bp CDP ?C(101-1516) and 330 bp peptide aptamers respectively, consistent with the expected results; We screened out peptide aptamers PEPTIDE 8 、32 、 46 and58 by BiFc experiments, which could against CDP ?C(101-1516); The result of co-immunoprecipitation technique revealed that the CDP ?C(101-1516) could be precipitated by screened peptide aptamers with FLAG tagged, indicated that there are interactions between CDP ?C(101-1516) and screened peptide aptamers.3.Expression and decision of the inhibitive effect of specific peptide aptamers: We obtained the lentivirus expression vector recombinant plasmid PCDH-PEPTIDE-GFP by high fidelity PCR and vector construction technology. After recombinant plasmids were identified by restriction endonuclease EcoRI and BamHI digestion analysis, the size of 330 bp peptide aptamers were obtained, consistent with the expected results; Green fluorescence was observed in 293 T cells under the fluorescence microscope after lentiviral packaging, suggest that the construction of lentivirus expression vectors were correctly; After infection with lentivirus supernatant, KYSE450 stable cell lines were obtained after drug selection, screened cells could express GFP indicated that the gene has been integrated into KYSE450 cells, we successfully obtained the esophageal squamous cell carcinoma cell line with the expression of peptide aptamers.4.The in vitro function assay showed that all stable cell lines have inhibitive roles in cell proliferation, and PEPTIDE 8 displayed the strongest inhibitive effect on proliferation, however, all these result require further demonstration. Conclusion1.We pinpointed the CDP-SOX2 binding domain:CDPΔC(101-1516);2.We screened out four peptide aptamers which could specifically bind to the CDPΔC(101-1516) domain: PEPTIDE 8, PEPTIDE 32, PEPTIDE 46 and PEPTIDE 58;3.We established several KYSE450 stable cell lines, a type of esophageal squamous cell carcinoma cell lines, to express specific peptide aptamers, including PEPTIDE 8, PEPTIDE 32,PEPTIDE 46 and PEPTIDE 58.4.Compared with the control, which is absent in the expression of peptide aptamer, PEPTIDE 8, PEPTIDE 32, PEPTIDE 46 and PEPTIDE 58 could inhibit the proliferation of KYSE450 cell in vitro, moreover, PEPTIDE 8 had the most significant inhibitory effect.