【作者】 赵燕英；

【导师】 陈正望；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 Daintain/AIF-1是近期发现的一种激素样新细胞因子,分子量为17kDa,含146个氨基酸残基,N端被乙酰化,主要由激活的巨噬细胞和T淋巴细胞表达、分泌,基因定位于人类白细胞抗原III区域,与器官移植排斥反应、自身性免疫疾病及肿瘤的发生发展等密切相关。为了进一步探讨Daintain/AIF-1的分布及功能,本研究以猪小肠为原料,通过稀醋酸低温浸提、海藻酸吸附、NaCl盐析、Sephadex G-25凝胶过滤和DEAE纤维素弱阴离子交换柱层析以及反相高效液相色谱等方法纯化得到蛋白纯品,离子化飞行质谱鉴定其分子量为16.692kDa,胰酶水解样品,并经反相高效液相色谱分离水解片断,取其中一片段进行氨基酸序列分析,其序列为YSSDEDLSRK,与Daintain/AIF-1分子内第36-45段残基完全一致,说明所得蛋白为Daintain/AIF-1。以制备的Daintain/AIF-1为免疫原免疫BALB/c小鼠,采用杂交瘤技术制备,酶联免疫吸附法(enzyme-linked immuno-sorbent assay,ELISA)筛选阳性杂交瘤细胞株,得到4株能稳定分泌抗体的单克隆细胞株(monoclonal antibodies,Mab)。利用ELISA试剂盒对单抗效价、亚类、亲和常数进行分析,4株单抗都属于IgG1,轻链为κ链。又经蛋白质印迹(Western blot)证实,所制备的抗体能特异性和Daintain/AIF-1结合,同时,竞争性抑制ELISA显示,标准Daintain/AIF-1可抑制制备的抗体与固相Daintain/AIF-1结合,进一步鉴定了Mab的特异性。这是国内外首次制备并鉴定出的抗Daintain/AIF-1单克隆抗体,可供器官移植排斥、自身免疫疾病和肿瘤诊断等相关学科研究之用。更多还原

【Abstract】 Daintain/allograft inflammatory factor 1 (AIF-1) is a 146-aa residue hormone-like cytokine discovered recently that is encoded within the HLA class III genomic region with a mass of 17kDa and an acetylated N terminus. Daintain/AIF-1 ,mainly expressed by activated macrophages and T lymphocytes,is involved in Transplant rejection, autoimmune disease and tumor progress etc.For further investigation of this peptide distribution and functions, Daintain/AIF-1 was isolated from porcine intestine with acetic acid extraction ,alginic acid adsorption, and sodium chloride saltingout followed by sephadex G-25(fine)column chromatography,DEAE anion exchange column chromatography and reverse-phase HPLC purification, The molecular mass of the fraction determined by MALDI-TOF-MS was 16.692kDa. After hydrolysised with trypsogen, the fragment separated by RP-HPLC was selected for sequence analysis as YSSDEDLSRK consensus with the 36-45 aa residue of Daintain/AIF-1, So the fraction identifed was Daintain/AIF-1.Daintain/AIF-1 purified was then used to immunize BALB/c mice as the immunogen. Furthermore, Both hybridization technique and enzyme linked immunosorbent assay(ELISA) were performed to generate cell lines which produced monoclonal antibodies(Mabs) against Daintain/AIF-1. Isotypes, titer and affinity of the Mabs belong to G1 subclass withκlight chain. Thereafter, the specificity of monoclonal antibodies was identified by Western blot and competitive inhibition ELISA.The Mabs against Daintain/AIF-1 prepared and identified in first time are useful tools for further investigation of transplant rejection, autoimmune diseases and cancer diagnosis.更多还原