本文關(guān)鍵詞：抗磷脂酰乙醇胺結(jié)合蛋白1（PEBP1）的鼠單克隆抗體的制備及應(yīng)用　出處：《南京醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： PEBP1 單克隆抗體 胰腺癌 Sandwich ELISA 組織芯片

【摘要】：磷脂酰乙醇胺結(jié)合蛋白1(phosphatidylethanolamine binding protein 1)即PEBP1,也稱Raf激酶抑制蛋白(raf kinase inhibitor protein, RKIP),屬于磷脂酰乙醇胺結(jié)合蛋白家族,廣泛存在于各種不同的生物中,參與了對細(xì)胞內(nèi)多種信號轉(zhuǎn)導(dǎo)通路的調(diào)節(jié)作用。PEBP1可以與Raf-1結(jié)合從而抑制MAPK信號轉(zhuǎn)導(dǎo)通路,并參與了對G蛋白偶聯(lián)受體信號通路和NF-κB信號通路的調(diào)控。PEBP1在膜的生物合成,精子發(fā)生,神經(jīng)發(fā)育和細(xì)胞凋亡等生理過程中發(fā)揮重要作用。2003年Fu et al發(fā)現(xiàn)PEBP1可以抑制腫瘤細(xì)胞轉(zhuǎn)移,使其成為抗腫瘤領(lǐng)域一個新的熱點。目前一致被認(rèn)為在前列腺癌,乳腺癌,結(jié)直腸癌,胃腸道間質(zhì)瘤,黑色素瘤,卵巢上皮癌中具有抑制腫瘤轉(zhuǎn)移的作用,并且是預(yù)后的標(biāo)志。但是在胰腺癌中的表達(dá)以及作用還沒有報道。本實驗室在前期工作中已經(jīng)獲得大量高純度的PEBP1蛋白標(biāo)準(zhǔn)品。本課題主要在于抗PEBP1的鼠單克隆抗體的制備及鑒定,雙抗體夾心ELISA方法的建立及研究其在胰腺癌組織中的應(yīng)用。本課題的主要內(nèi)容及設(shè)計如下: 1.抗PEBP1抗體的制備及初步鑒定。 2.雙抗體夾心ELISA方法的建立 3.抗PEBP1抗體在胰腺癌組織中的應(yīng)用。 材料與方法 1.抗PEBP1抗體的制備及初步鑒定: 以純化的PEBP1蛋白免疫BALB/c小鼠,之后取脾細(xì)胞使之與骨髓瘤細(xì)胞OUR-1融合。隨后通過ELISA篩選陽性雜交瘤,經(jīng)過三輪的亞克隆,選擇4個OD值最高,并且有不同活性的克隆,使用Protein G HP純化獲得抗體。獲得抗體之后,進(jìn)行抗體的初步鑒定,選擇高表達(dá)和低表達(dá)該蛋白的人胰腺癌細(xì)胞系,通過Western Blot鑒定該組抗體識別該蛋白的特異性。將GFP-PEBP1轉(zhuǎn)染到NIH3T3細(xì)胞中,將其固定,用所制備的抗體進(jìn)行免疫熒光實驗。 2.雙抗體夾心ELISA方法的建立 通過ForteBio’s Octet系統(tǒng)選擇1對與該蛋白結(jié)合力高的抗體做Sandwich ELISA,可以用于進(jìn)一步快速早期檢測和診斷胰腺癌。 3.抗PEBP1抗體在胰腺癌組織中的應(yīng)用 PEBP1被認(rèn)為是抑制腫瘤轉(zhuǎn)移的基因,我們在此將抗體用于組織芯片,研究PEBP1在胰腺癌病人和正常人中的表達(dá)是否不同。 研究結(jié)果 1.成功獲得四株單克隆抗體,分別有不同的活性。7F12在Western Blot中可以識別正常的293細(xì)胞和部分胰腺癌細(xì)胞中的PEBP1. 2.由ForteBio’s Octet系統(tǒng)篩選出一對抗體:4F10和8E2,可以識別PEBP1蛋白的不同的表位,因此這一對抗體可以用于Sandwich ELISA,它們可以檢測出PEBP1在ng/ml的水平,經(jīng)過優(yōu)化之后可以用于臨床實踐中測定病人血清,可以進(jìn)一步快速早期檢測和診斷胰腺癌。 3.單克隆抗體4A11在組織芯片中可以檢測到在正常的胰腺組織中的PEBP1。而在胰腺癌組織中,PEBP1的表達(dá)很低甚至沒有。 結(jié)論 我們的結(jié)果顯示PEBP1在正常的胰腺中高表達(dá),而在胰腺癌組織中的表達(dá)明顯降低或者消失�？筆EBP1的單克隆抗體4A11, 4F10, 7F12和8E2是潛在的臨床診斷胰腺癌的試劑。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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本文編號：1361114