人CD55抗原模拟表位的筛选及其对乳腺癌细胞的抑瘤作用研究
发布时间：2019-05-06 17:32
【摘要】：目的:利用噬菌体肽库展示技术从Ph.D.12噬菌体肽库中筛选能够和人CD55单克隆抗体(CD55 Mc Ab)特异性结合的短肽,从中找到CD55的抗原模拟表位,设计出与人肿瘤免疫逃逸蛋白CD55同源的的短肽,为肿瘤治疗奠定理论基础。方法:以人CD55单克隆抗体为靶标,Ph.D.12肽库通过4轮噬菌体筛选淘选出与CD55单克隆抗体特异性结合的短肽,计算每轮回收率。4轮筛选后,随机挑选11个噬菌体单克隆和野生型噬菌体进行竞争结合,计算其结合力。利用ELISA、竞争结合实验鉴定筛选出结合力强的噬菌体单克隆,E.coli ER2378宿主菌表达和纯化噬菌体。将阳性噬菌体克隆交由公司测序,测序结果里找出出现频率高的短肽序列,设计与人CD55单克隆抗体具有高亲和性及特异性的短肽,并对其体外抗乳腺癌作用效果进行研究。CCK8实验检测CD55抗原模拟表位对乳腺癌细胞(MDA-MB-231和MCF-7细胞)增殖的影响。荧光显微镜验证短肽在细胞中的摄入和分布。透射电镜(Transmission electron microscopy,TEM)和流式细胞术检测该短肽在乳腺癌细胞MDA-MB-231细胞和MCF-7细胞凋亡中的影响。结果:4轮筛选后,每轮噬菌体回收率都达到了较高数量级。竞争结合实验结果显示11个噬菌体单克隆和第3、4轮筛选后的噬菌体都与CD55单克隆抗体亲和力都非常显著。ELISA结果显示有8个噬菌体单克隆和CD55单克隆抗体亲和力显著。根据测序结果得到两条高表达短肽序列HAHTPTRGVMHA(简称H肽)和QVNGLGERSQQM(简称Q肽)。CCK8实验证明Q短肽序列对乳腺癌MDA-MB-231细胞和MCF-7细胞的毒性作用显著且具有剂量依赖性,而H短肽较Q肽药效较差,达到相同抑瘤率所需作用浓度较高。荧光显微镜观察到Q短肽分布在细胞膜表面。透射电镜和流式细胞术显示Q短肽具有诱导MDA-MB-231和MCF-7细胞凋亡的作用。结论:利用噬菌体随机12肽库成功得到两条CD55单克隆抗体特异性结合的CD55抗原模拟表位短肽序列HAHTPTRGVMHA和QVNGLGERSQQM,其中Q肽比H肽作用更显著,能够抑制乳腺癌细胞增殖并诱导其凋亡,为乳腺癌靶向治疗提供新思路和新靶标。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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