人CD55抗原模擬表位的篩選及其對(duì)乳腺癌細(xì)胞的抑瘤作用研究
[Abstract]:Objective: to screen short peptides that can specifically bind to human CD55 monoclonal antibody (CD55 Mc Ab) from Ph.D.12 phage peptide library by phage peptide library display technique, and to find the epitope of CD55 antigen. A short peptide homologous to human tumor immune escape protein CD55 was designed to lay a theoretical foundation for tumor therapy. Methods: using human CD55 monoclonal antibody as target, Ph.D.12 peptide library was selected by four rounds of phage screening to select short peptides specific to CD55 monoclonal antibody. The recovery rate of each round was calculated. After 4 rounds of screening, a short peptide binding to CD55 monoclonal antibody was selected. Eleven phage clones and wild bacteriophages were randomly selected for competitive binding and their binding capacity was calculated. ELISA, competitive binding assay was used to identify and screen phage monoclonal, E.coli ER2378 host bacteria and pure bacteriophage. The positive phage clone was transferred to the company for sequencing. The sequence of the short peptide with high frequency was found in the sequencing results, and the short peptide with high affinity and specificity with the monoclonal antibody against human CD55 was designed. The effects of CD55 mimotope on the proliferation of breast cancer cells (MDA-MB-231 and MCF-7 cells) were detected by CCK8 assay. Fluorescence microscopy was used to verify the uptake and distribution of short peptides in cells. Transmission electron microscopy (Transmission electron microscopy,TEM) and flow cytometry (FCM) were used to detect the effect of the peptide on the apoptosis of breast cancer cells MDA-MB-231 cells and MCF-7 cells. Results: after 4 rounds of screening, the recovery rate of bacteriophage in each round reached a higher order of magnitude. The results of competitive binding assay showed that the affinity of 11 phage clones and 3 and 4 rounds of screening bacteriophages to CD55 monoclonal antibodies were very significant. Elisa results showed that 8 phage clones and CD55 monoclonal antibodies had significant affinity. According to the sequencing results, two highly expressed short peptide sequences, HAHTPTRGVMHA (H peptide) and QVNGLGERSQQM (Q peptide), were obtained. The results of CCK8 assay showed that the toxicity of Q peptide sequence to breast cancer cell line MDA-MB-231 and MCF-7 cells was significant and dose dependent. The effect of H-peptide was worse than that of Q-peptide, and the effect concentration of H-peptide was higher than that of Q-peptide to achieve the same tumor inhibition rate. Q-short peptide was observed on the surface of cell membrane by fluorescence microscope. Transmission electron microscopy (TEM) and flow cytometry (FCM) showed that Q-peptide could induce apoptosis of MDA-MB-231 and MCF-7 cells. Conclusion: two peptide sequences of CD55 antigen mimetic epitope HAHTPTRGVMHA and QVNGLGERSQQM, specifically binding to two monoclonal antibodies against CD55 were successfully obtained by using phage random 12 peptide library. Q peptide was more effective than H peptide, which could inhibit the proliferation and induce apoptosis of breast cancer cells. It provides a new idea and a new target for breast cancer targeted therapy.
【學(xué)位授予單位】:青島大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2017
【分類號(hào)】:R737.9
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