【摘要】：目的:利用噬菌體肽庫展示技術(shù)從Ph.D.12噬菌體肽庫中篩選能夠和人CD55單克隆抗體(CD55 Mc Ab)特異性結(jié)合的短肽,從中找到CD55的抗原模擬表位,設(shè)計(jì)出與人腫瘤免疫逃逸蛋白CD55同源的的短肽,為腫瘤治療奠定理論基礎(chǔ)。方法:以人CD55單克隆抗體為靶標(biāo),Ph.D.12肽庫通過4輪噬菌體篩選淘選出與CD55單克隆抗體特異性結(jié)合的短肽,計(jì)算每輪回收率。4輪篩選后,隨機(jī)挑選11個(gè)噬菌體單克隆和野生型噬菌體進(jìn)行競爭結(jié)合,計(jì)算其結(jié)合力。利用ELISA、競爭結(jié)合實(shí)驗(yàn)鑒定篩選出結(jié)合力強(qiáng)的噬菌體單克隆,E.coli ER2378宿主菌表達(dá)和純化噬菌體。將陽性噬菌體克隆交由公司測序,測序結(jié)果里找出出現(xiàn)頻率高的短肽序列,設(shè)計(jì)與人CD55單克隆抗體具有高親和性及特異性的短肽,并對(duì)其體外抗乳腺癌作用效果進(jìn)行研究。CCK8實(shí)驗(yàn)檢測CD55抗原模擬表位對(duì)乳腺癌細(xì)胞(MDA-MB-231和MCF-7細(xì)胞)增殖的影響。熒光顯微鏡驗(yàn)證短肽在細(xì)胞中的攝入和分布。透射電鏡(Transmission electron microscopy,TEM)和流式細(xì)胞術(shù)檢測該短肽在乳腺癌細(xì)胞MDA-MB-231細(xì)胞和MCF-7細(xì)胞凋亡中的影響。結(jié)果:4輪篩選后,每輪噬菌體回收率都達(dá)到了較高數(shù)量級(jí)。競爭結(jié)合實(shí)驗(yàn)結(jié)果顯示11個(gè)噬菌體單克隆和第3、4輪篩選后的噬菌體都與CD55單克隆抗體親和力都非常顯著。ELISA結(jié)果顯示有8個(gè)噬菌體單克隆和CD55單克隆抗體親和力顯著。根據(jù)測序結(jié)果得到兩條高表達(dá)短肽序列HAHTPTRGVMHA(簡稱H肽)和QVNGLGERSQQM(簡稱Q肽)。CCK8實(shí)驗(yàn)證明Q短肽序列對(duì)乳腺癌MDA-MB-231細(xì)胞和MCF-7細(xì)胞的毒性作用顯著且具有劑量依賴性,而H短肽較Q肽藥效較差,達(dá)到相同抑瘤率所需作用濃度較高。熒光顯微鏡觀察到Q短肽分布在細(xì)胞膜表面。透射電鏡和流式細(xì)胞術(shù)顯示Q短肽具有誘導(dǎo)MDA-MB-231和MCF-7細(xì)胞凋亡的作用。結(jié)論:利用噬菌體隨機(jī)12肽庫成功得到兩條CD55單克隆抗體特異性結(jié)合的CD55抗原模擬表位短肽序列HAHTPTRGVMHA和QVNGLGERSQQM,其中Q肽比H肽作用更顯著,能夠抑制乳腺癌細(xì)胞增殖并誘導(dǎo)其凋亡,為乳腺癌靶向治療提供新思路和新靶標(biāo)。
[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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