【作者】 黄宏亮；

【导师】 周红；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 目的:获得高纯度人血浆β2糖蛋白I（β2GPI）,制备兔抗人β2GPI多克隆抗体,为进一步研究β2GPI及其抗体在抗磷脂综合征（APS）病理机制中的作用奠定基础。方法:1.采用HClO₄沉淀、Heparin Sepharose CL-6B亲和层析等方法纯化人血浆β2GPI;利用SDS-PAGE电泳、免疫印迹分析提纯蛋白的纯度;观察不同温度、NaCl浓度及防腐剂等条件下的β2GPI稳定性。2.以纯化的β2GPI作为抗原免疫新西兰大白兔,获得抗血清;进而利用Protein A及β2GPI亲和层析柱纯化抗血清中的总IgG及抗β2GPI抗体;采用免疫双向扩散试验（IDD）、ELISA和免疫印迹等方法进行抗体验证。3.应用人β2GPI及其多克隆抗体共同刺激单核细胞株THP-1,检测其组织因子（TF）活性表达情况;利用β2GPI亲和层析柱,初步分析β2GPI与单核细胞株THP-1表面膜联蛋白A2（Annexin A2）的结合情况。结果:1.本法获得了高纯度目的蛋白β2GPI（＞95%）。β2GPI在低于0.8 mol/L NaCl溶液中室温及4℃保存易降解变性,但在1.6mol/L NaCl溶液中室温及4℃保存60天均未见蛋白明显降解。2.IDD及ELISA鉴定抗血清及总IgG效价分别达到1:16和1:100000;免疫印迹显示纯化的总IgG和抗β2GPI抗体与β2GPI蛋白均有特异反应条带;3.β2GPI及其多克隆抗体形成的复合物能够刺激单核细胞株THP-1表达TF活性;免疫印迹实验表明,来自THP-1细胞的Annexin A2能够与β2GPI亲和柱结合。结论:1.通过本研究,简单方便地获得了高纯度人血浆β2GPI;在使用和保存β2GPI时要考虑到其降解的可能性,注意保存条件,必要时冻干保存。2.通过免疫动物及亲和层析方法,成功获得了兔抗人β2GPI多克隆抗体。3.应用制备的β2GPI亲和层析柱,证明了β2GPI可以结合单核细胞株THP-1表面的Annexin A2;β2GPI与其抗体复合物能够刺激THP-1细胞表达TF活性,是导致APS高凝状态的重要机理之一。更多还原

【Abstract】 Objective:To purifyβ2-glycoprotein I（β2GPI） from normal human plasma and prepare rabbit polyclonal antibodies against humanβ2GPI for establishing the foundation of the further study on the roles of anti-β2GPI/β2GPI in the pathology of antiphospholipid syndrome（APS）.Methods:1.β2GPI was purified by Heparin Sepharose CL-6B affinity chromatography following by HClO₄ precipitation.The purity of the isolatedβ2GPI was identified by SDS-PAGE,Western blotting（WB） analysis.And it’s stability was observed under different temperature, concentration of salt and antiseptics.2.The New Zealand White rabbit was immunized with purified humanβ2GPI and the anti-serum was made. The whole IgG and its anti-β2GPI antibody in the anti-serum were purified by Protein A andβ2GPI-affinity column.The specificity and sensitivity of the antibody were identified by IDD,ELISA and WB.3. The monocytic cell line THP-1 cells were treated withβ2GPI and its polyclonal antibodies,and the TF activity in the cells was detected.The binding of Annexin A2 in the THP-1 cell lysates toβ2GPI was analyzed by theβ2GPI-affinity column.Results:1.β2GPI was harvested using above methods and its purity was confirmed more than 95%.The protein maintained in≤0.8 mol/L of NaCl might be degraded at room temperature,even though at 4℃.But it was not obviously degraded if it was maintained in 1.6 mol/L of NaCl at room temperature and 4℃.2.The reactive titers of the anti-serum and the whole IgG reached to 1:16 and 1:100000 with IDD and ELISA respectively.Both the whole IgG and its anti-β2GPI antibody could react withβ2GPI by WB.3.The complex ofβ2GPI and its antibody could induce the TF activity expressed on monocytic cell line THP-1.And the Annexin A2 in the THP-1 cell lysates could bind toβ2GPI-affinity column.Conclusions:1.Humanβ2GPI with high purity from the plasma can be obtained by our methods.It is important to protectβ2GPI from denaturazation in the process of conservation and regular use.It is the best way to dry the protein into powder for keepingβ2GPI long time.2. The polyclonal antibodies against humanβ2GPI can be successfully prepared by the immunization of rabbit withβ2GPI and the purification by chromatography.3.β2GPI can bind to the Annexin A2 existed in the monocytes,which has been analyzed byβ2GPI-affinity column.β2GPI and its antibodies can enhance the TF activity on monocytes,which is contributed to the mechanism of hypercoagulability in APS.更多还原