抗磷脂酰乙醇胺结合蛋白1（PEBP1）的鼠单克隆抗体的制备及应用

发布时间：2017-12-31 19:19

本文关键词：抗磷脂酰乙醇胺结合蛋白1（PEBP1）的鼠单克隆抗体的制备及应用　出处：《南京医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：磷脂酰乙醇胺结合蛋白1(phosphatidylethanolamine binding protein 1)即PEBP1,也称Raf激酶抑制蛋白(raf kinase inhibitor protein, RKIP),属于磷脂酰乙醇胺结合蛋白家族,广泛存在于各种不同的生物中,参与了对细胞内多种信号转导通路的调节作用。PEBP1可以与Raf-1结合从而抑制MAPK信号转导通路,并参与了对G蛋白偶联受体信号通路和NF-κB信号通路的调控。PEBP1在膜的生物合成,精子发生,神经发育和细胞凋亡等生理过程中发挥重要作用。2003年Fu et al发现PEBP1可以抑制肿瘤细胞转移,使其成为抗肿瘤领域一个新的热点。目前一致被认为在前列腺癌,乳腺癌,结直肠癌,胃肠道间质瘤,黑色素瘤,卵巢上皮癌中具有抑制肿瘤转移的作用,并且是预后的标志。但是在胰腺癌中的表达以及作用还没有报道。本实验室在前期工作中已经获得大量高纯度的PEBP1蛋白标准品。本课题主要在于抗PEBP1的鼠单克隆抗体的制备及鉴定,双抗体夹心ELISA方法的建立及研究其在胰腺癌组织中的应用。本课题的主要内容及设计如下: 1.抗PEBP1抗体的制备及初步鉴定。 2.双抗体夹心ELISA方法的建立 3.抗PEBP1抗体在胰腺癌组织中的应用。材料与方法 1.抗PEBP1抗体的制备及初步鉴定: 以纯化的PEBP1蛋白免疫BALB/c小鼠,之后取脾细胞使之与骨髓瘤细胞OUR-1融合。随后通过ELISA筛选阳性杂交瘤,经过三轮的亚克隆,选择4个OD值最高,并且有不同活性的克隆,使用Protein G HP纯化获得抗体。获得抗体之后,进行抗体的初步鉴定,选择高表达和低表达该蛋白的人胰腺癌细胞系,通过Western Blot鉴定该组抗体识别该蛋白的特异性。将GFP-PEBP1转染到NIH3T3细胞中,将其固定,用所制备的抗体进行免疫荧光实验。 2.双抗体夹心ELISA方法的建立通过ForteBio’s Octet系统选择1对与该蛋白结合力高的抗体做Sandwich ELISA,可以用于进一步快速早期检测和诊断胰腺癌。 3.抗PEBP1抗体在胰腺癌组织中的应用 PEBP1被认为是抑制肿瘤转移的基因,我们在此将抗体用于组织芯片,研究PEBP1在胰腺癌病人和正常人中的表达是否不同。研究结果 1.成功获得四株单克隆抗体,分别有不同的活性。7F12在Western Blot中可以识别正常的293细胞和部分胰腺癌细胞中的PEBP1. 2.由ForteBio’s Octet系统筛选出一对抗体:4F10和8E2,可以识别PEBP1蛋白的不同的表位,因此这一对抗体可以用于Sandwich ELISA,它们可以检测出PEBP1在ng/ml的水平,经过优化之后可以用于临床实践中测定病人血清,可以进一步快速早期检测和诊断胰腺癌。 3.单克隆抗体4A11在组织芯片中可以检测到在正常的胰腺组织中的PEBP1。而在胰腺癌组织中,PEBP1的表达很低甚至没有。结论我们的结果显示PEBP1在正常的胰腺中高表达,而在胰腺癌组织中的表达明显降低或者消失。抗PEBP1的单克隆抗体4A11, 4F10, 7F12和8E2是潜在的临床诊断胰腺癌的试剂。
[Abstract]:Phosphatidylethanolamine binding protein 1 (PEBP1). Also known as Raf kinase inhibitor protein kinase inhibitor protein, RKIPP belongs to the phosphatidylethanolamine binding protein family. In a variety of organisms, involved in the regulation of a variety of intracellular signal transduction pathways. PEBP1 can bind to Raf-1 to inhibit the MAPK signal transduction pathway. And involved in the G protein coupled receptor signaling pathway and NF- 魏 B signal pathway regulation. PEBP1 in the membrane biosynthesis, spermatogenesis. In 2003, Fu et al found that PEBP1 can inhibit the metastasis of tumor cells. It has become a new hot spot in the field of antitumor. It is widely considered that it has the role of inhibiting tumor metastasis in prostate cancer, breast cancer, colorectal cancer, gastrointestinal stromal tumor, melanoma, ovarian epithelial carcinoma. And it is a prognostic marker. However, the expression and role in pancreatic cancer have not been reported. In our laboratory, a large number of high purity PEBP1 protein standard products have been obtained in previous work. The main purpose of this study is to resist PE. Preparation and identification of mouse monoclonal antibody against BP1. Establishment and Application of double Antibody Sandwich ELISA method in Pancreatic Cancer. The main contents and designs of this study are as follows: 1. Preparation and preliminary identification of anti-PEBP1 antibody. 2. Establishment of double antibody sandwich ELISA method 3. Application of anti PEBP1 antibody in pancreatic carcinoma. Materials and methods 1. Preparation and preliminary identification of anti PEBP1 antibody: The purified PEBP1 protein was used to immunize the BALB/c mice, and then the spleen cells were fused with the myeloma cell OUR-1. Then the hybridoma were screened by ELISA. After three rounds of subcloning, four clones with the highest OD value and different activity were selected. The antibody was purified by Protein G HP. After the antibody was obtained, the antibody was preliminarily identified. Human pancreatic cancer cell lines with high and low expression of the protein were selected. The antibody was identified by Western Blot to identify the specificity of the protein. GFP-PEBP1 was transfected into NIH3T3 cells and immobilized. Immunofluorescence assay was carried out with the prepared antibody. 2. Establishment of double antibody sandwich ELISA method A pair of antibodies with high binding ability to the protein was selected for Sandwich ELISA by ForteBio's Octet system. Can be used for further rapid early detection and diagnosis of pancreatic cancer. 3. Application of anti PEBP1 antibody in pancreatic carcinoma PEBP1 is considered to be a gene that suppresses tumor metastasis. We use the antibody in tissue microarray to study whether the expression of PEBP1 in pancreatic cancer patients is different from that in normal controls. Research results 1. Four monoclonal antibodies were successfully obtained. Different activity of. 7F12 can recognize PEBP1 in normal 293 cells and some pancreatic cancer cells in Western Blot. 2. A pair of antibodies: 4F10 and 8E2 were screened by ForteBio's Octet system, which could recognize different epitopes of PEBP1 protein. Therefore, this pair of antibodies can be used in Sandwich Elsa, they can detect the level of PEBP1 in ng/ml, after optimization can be used to determine the patient's serum in clinical practice. It can be used to detect and diagnose pancreatic cancer quickly. 3.Monoclonal antibody 4A11 could detect PEBP1in normal pancreatic tissue in tissue microarray, but the expression of PEBP1 in pancreatic carcinoma was very low or not. Conclusion Our results showed that PEBP1 was highly expressed in normal pancreas, but decreased or disappeared in pancreatic carcinoma. Monoclonal antibody 4A11,4F10 against PEBP1. 7 F 12 and 8 E 2 are potential clinical diagnostic agents for pancreatic cancer.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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