环加氧酶2启动子区抑制性结合蛋白Nono的原核表达和多克隆抗体的制备-生物化学与分子生物学专业论文.docxVIP

南京医科大学硕士学位论文环加氧酶_2启动子区抑制性结合蛋白Nono的原核表达和多 南京医科大学硕士学位论文 环加氧酶_2启动子区抑制性结合蛋白Nono的原核表达和多 克隆抗体的制备 摘要 环加氧酶-2(cyclooxygenase·2，COX-2)是体内催化产生前列腺素 (PGs)等炎症活性物质的关键酶。作为一种诱导型合酶，COX．2可被 细胞因子、生长因子、有丝分裂原等多种刺激因素诱导表达。研究已 证实，COX．2的异常高表达与炎症、肿瘤、心血管疾病以及糖尿病等 多种疾病的发生发展密切相关． 本实验室在前期工作中发现，小鼠COX．2启动子区一655／-632位置 上存在着一个转录抑制元件，并且质谱鉴定提示与该元件相结合的其 中一种DNA结合蛋白 Nono(non-POU-domain-eontaining．octamer- binding protein)。Nono是．．--种功能复杂的核蛋白，参与细胞核内RNA 加工、基因的转录调控、DNA重组配对等多个核事件过程。为了在前 期电泳迁移率改变实验的基础上，进一步马奎证Nono蛋白确实与此抑 制性序列结合，本．课题拟在大肠杆菌中表达Nono蛋白，作为抗原免 疫小鼠制#Nono的多克隆抗体，用于超迁移实验。 结果显示，pET-28a(+)-Nono重组表达质粒构建成功，在大肠杆 菌中诱导表达出的His—Nono融合蛋白达到了作为抗原免疫小鼠的要 求。最终获得了滴度较高，特异性较好的．抗Nono抗血清，纯化出的 抗Nono多克隆抗体不仅用于超迁移实验，也为以后进一步研究Nono 蛋白在COX．2转录调控方面所起的作用打下基础． 南京医科大学硕士学位论文【关键词】COX．2基因； 南京医科大学硕士学位论文 【关键词】COX．2基因； 基因重组；融合蛋白；多克隆抗体 2 南京医科大学硕士学位论文Prokaryotic 南京医科大学硕士学位论文 Prokaryotic Expression of Nono，a Novel DNA Bingding Protein of the Repressor Element in the CycloOxygenase-2 Promoter,and Preparation of its Polyclonal Antibody Abstract Cyclooxygenase一2(cox一21 is a key enzyme that catalyzes the synthesis of prostaglandins．It is not constitutively expressed but can be greatly induced by cytokines，growth factors and mitogens，et a1．It has been demonstrated that the abnormal overexpression of COX02 plays all important role in inflammation，carcinogenesis，cardiovascular diseases and the development of diabetes． In the prior studies，we report for the first time a novel repressor element located between nucleotides．655／．632 of the mouse COX．2 promoter．One of the nuclear proteins binding to this element Was identified as a non—POU-domain—containing，octamer-binding protein (Nono)．Nono exhibits mulfi-ftmctional characteristics in a variety of nuclear processes，such as RNA processing，transcriptional regulation and DNA unwinding．To confirm the binding of Nono to the repressor element，the polyclonal antibody against Nono Was prepared for the antibody—based supershift assay． The resuks suggested that the recombinant prokaryotic expression vector pET-28a(