重组细胞生长因子结构与功能关系的研究

发布时间：2018-08-02 09:02

【摘要】：[研究背景] 人促血液血管细胞生长因子(Hemangiopoietin，HAPO)是我室研究发现的一个新的细胞生长因子，具有促进造血干细胞，内皮祖细胞增殖、黏附、趋化的功能。HAPO蛋白最初是从再生障碍性贫血病人尿液中提取的天然蛋白，我室对其功能进行了深入的研究后成功地构建了原核表达体系，并测定了重组蛋白的细胞因子活性。 N端测序显示HAPO蛋白是糖蛋白4(proteoglycan 4，PRG4)的一个不同剪接体形式。HAPO蛋白N端为由PRG4第2、3外显子编码的两个促细胞生成素B结构域(Somatomedin B，SMB)，中间为第4外显子编码的假定的肝素结合区(putative Heparin Binding Domain，pHBD)，C端为第6外显子编码的黏液素样重复序列。PRG4尚有若干剪接体形式，其中有两种为第4、5外显子同时缺失和3、4、5三个外显子同时缺失。这为表达缺失突变蛋白以研究rhHAPO各个结构域的功能提供了条件。目前国内外对HAPO蛋白的各项研究课题主要集中在本实验室以及与香港和法国等实验室的合作上，工作的重点在于临床实验探讨HAPO与疾病的相关性，体内外实验探讨HAPO生理功能等。已制备得到单克隆抗体，钓得受体，，发现下游信号传导途径之一为磷酸酯酶3激酶ATP途径等等，但是对于其结构的研究是一个空白。仅仅局限于NCBI网站BLAST数据库搜索程序给出的关于HAPO蛋白含有4个结构域的信息。因此，rhHAPO结构和功能相结合的研究将能够更好的理解与HAPO蛋白相关的各种实验现象、实验结果。 [实验内容] 本研究在分子克隆构建原核表达体系基础上，利用HPLC，MALDI-TOF-MS，CD，DLS等实验技术研究了以下四个版块的内容，彼此之间有交叉和衔接：单体分子四个结构域的结构特点对功能发挥的影响；不同聚集体分子功能的比较；不同剪接体蛋白对聚集行为的影响；验证假定的肝素结合区(pHBD)是否结合肝素。 [实验目的] 阐明rhHAPO活性功能区所在；阐明形成聚集的区域所在；找到活性最高稳定性最佳的剪接体和聚集体形式；提供初步的理化特性，为rhHAPO新药的研发提供实验室数据。
[Abstract]:[background] Hemangiopoietine HAPO (HAPO) is a new cell growth factor found in our laboratory. It can promote the proliferation and adhesion of hematopoietic stem cells and endothelial progenitor cells. The function of chemotaxis. HAPO protein was originally extracted from the urine of aplastic anemia patients. The N-terminal sequencing showed that HAPO protein was a different splice form of proteoglycan 4 (proteoglycan 4 / PRG4). HAPO protein N The two cytopoietin B domains (Somatomedin BnSMB) encoded by exon 2 of PRG4, and the assumed heparin binding region (putative Heparin Binding Domainn pHBD) at the middle of exon 4, have myxin-like repeats at the C end of exon 6. PRG4 still exists. Several splicing forms, Two of them were simultaneous deletion of exon 4 and 3 exon 3. This provides conditions for the expression of deletion mutant proteins to study the function of rhHAPO domains. At present, the research topics of HAPO protein at home and abroad are mainly focused on our laboratory and the cooperation with Hong Kong and France. The focus of our work is clinical experiments to explore the correlation between HAPO and disease. To investigate the physiological function of HAPO in vivo and in vitro. Monoclonal antibodies were prepared, receptors were isolated, and one of the downstream signal transduction pathways was found to be phosphatase 3 kinase ATP pathway, but the study of its structure is a blank. It is only limited to the information given by the BLAST database searcher of NCBI website that HAPO protein contains four domains. Therefore, the research on the combination of structure and function of rhHAPO will be able to better understand the various experimental phenomena related to HAPO protein and the experimental results. [experimental contents] based on the construction of prokaryotic expression system by molecular cloning, the contents of the following four plates were studied by using HPLCU MALDI-TOF-MSMSCD DLS and other experimental techniques. The structure characteristics of the four domains of monomers affect the function, the comparison of the functions of different aggregates, the effects of different splicing proteins on the aggregation behavior; To verify whether the assumed heparin binding region (pHBD) binds heparin. [objective] to elucidate the active functional region of rhHAPO, to clarify the region where the aggregation is formed, to find the best splicing and aggregate form with the highest activity and stability, to provide the preliminary physicochemical properties and to provide laboratory data for the development of new drugs for rhHAPO.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

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