ⅱ型跨膜丝氨酸蛋白酶hatl4表达及其单克隆抗体研制-expression of type ii transmembrane serine protease hat 4 and preparation of its monoclonal antibody.docx

II 型跨膜丝氨酸蛋白酶 HATL4 的表达及其单克隆抗体的研制中文摘要有 1.23%。免疫荧光发现我们研制的抗体能特异性识别 HATL4，并发现 HATL4 定位于肿瘤细胞膜表面。结论：本研究首次发现 HATL4 在急性髓细胞白血病中表达增高；成功表达、纯 化和复性了 HATL4 融合蛋白；并运用杂交瘤技术成功筛选并制备出 5 株抗 HATL4 的单克隆抗体细胞株；经蛋白质免疫印迹法、流式细胞术和免疫荧光鉴定，制备的 5 株单克隆抗体均能特异性识别 HATL4 蛋白。关键词：肿瘤；恶性血液疾病；HATL4；蛋白酶；II 型跨膜丝氨酸蛋白酶；单克 隆抗体；作者：蒋艺枝 指导老师：董宁征英文摘要II 型跨膜丝氨酸蛋白酶 HATL4 的表达及其单克隆抗体的研制Expression and Characterization of Type II Transmembrane Serine Protease HATL4 and ItsMonoclonal AntibodiesAbstractObjective: The type II transmembrane serine protease (TTSPs) family includes a group of trypsin-like enzymes that are anchored on the cell surface via an integral transmembrane domain near the N-terminus. Many TTSPs have been found to play an important role in a variety of physiological and pathological processes. Dysregulation of these TTSPs may lead to serious diseases such as cancer, anemia, hearing loss, and cardiovascular disease. There are 17 TTSP members in humans. The human airway trypsin-like protease, HAT, was originally detected in the extracellular airway fluids of patients with chronic airway diseases, which plays a potential role in mucilage production, hydrolysis of hemagglutinin antigen of flu virus, cell migration and degradation of basilar membrane. To date, however, there is little information regarding the physiological functions of HATL4, a member of HAT/DESC subfamily. The goal of this study is to examine the expression of HATL4 in leukemia cell lines and hematological malignancies and expression and purification of HATL4 recombinant protein to produce monoclonal antibody against HATL4.Methods:RT-PCR was used to analyze HATL4 mRNA expression in hematological cancer cell lines and bone marrow cells from hematological malignancies.The gene fragment encoding human HATL4 protease domain was amplified from the human leukemia cell line MEG-01 by RT-PCR. After gel extracting, the gene was cloned into pMD18-T simple vector and transformed to E.coli Top 10. After identification by restriction endonuclease digestion