β_2糖蛋白1多肽抗体的制备与应用

Preparation and application of a polyclonal antibody against the peptides of β_2 glycoprotein 1

目的 利用人工合成β2糖蛋白1（β2GP1）多肽制备针对人源、鼠源β2GP1的多克隆抗体, 并鉴定抗体的特异性及致病性。方法 应用Fmoc法化学合成β2GP1 N端第35~51位氨基酸的多肽, 将合成后的多肽与钥孔血蓝蛋白（KLH）偶联, 免疫新西兰大白兔制备抗血清, 蛋白G纯化得到抗β2GP1多肽抗体, 利用ELISA和Western blot法鉴定其效价和特异性。体外实验使用抗β2GP1多肽抗体/β2GP1复合物刺激C3H/HeN小鼠腹腔巨噬细胞一定时间, 收集细胞总RNA和总蛋白, 实时定量PCR、Western blot法分别检测细胞组织因子（TF）mRNA和蛋白表达; Western blot法检测细胞p38、磷酸化p38、核因子κB p65（NF-κB p65）及磷酸化NF-κB p65表达情况; 体内实验采用C3H/HeN小鼠腹腔注射抗β2GP1多肽抗体建立实验性抗磷脂综合征（EAPS）模型, 检测小鼠外周血抗β2GP1抗体滴度及部分凝血活酶时间（APTT）。结果 化学合成β2GP1多肽的纯度为94％, 达到免疫用抗原标准。偶联KLH后免疫新西兰大白兔, 其抗血清效价大于1∶32 000。Western blot结果显示抗β2GP1多肽抗体可特异性识别人源和鼠源β2GP1条带, 双抗夹心ELISA检测表明该多肽抗体可与β2GP1隐蔽表位特异性结合。体外实验显示抗β2GP1多肽抗体/β2GP1复合物能够增强小鼠腹腔巨噬细胞p38、 NF-κB p65磷酸化, 诱导细胞TF mRNA及蛋白表达。体内实验成功建立EAPS小鼠模型, 小鼠外周血抗β2GP1抗体滴度大于1∶3200, APTT明显缩短。结论 所制备的抗β2GP1多肽抗体可识别人源和鼠源β2GP1分子, 能与β2GP1隐蔽抗原特异性结合且具有致病效应。

Objective To prepare a polyclonal antibody against human and murine β2 glycoprotein 1 （β2GP1） antigen with chemically synthesized β2GP1 peptides, and identify its specificity and pathogenicity. Methods The peptides from the NH2-terminal 35th-51th amino acids of β2GP1 were synthesized by standard Fmoc assay, and then used to immunize New Zealand white rabbits after coupling with keyhole limpet hemocyanin （KLH）. The polyclonal antibody in the rabbit sera was purified by protein G column. The titer and specificity of the polyclonal antibody were determined by ELISA and Western blot analysis. The total RNA was extracted and the protein lysates were collected from C3H/HeN mouse peritoneal macrophages treated with the above anti-β2GP1 peptides antibody/β2GP1 complexes in vitro. And the tissue factor （TF） mRNA and protein expression in the peritoneal macrophages were detected by real-time quantitative PCR and Western blotting. The activation of p38 and NF-κB 1065 induced by anti-β2GP1 peptides antibody/β2GP1 complexes was determined by Western blotting using phosphor-specific antibodies. Experimental antiphospholipid antibody syndrome （EAPS） mouse model was established in C3H/HeN mice by intraperitoneal injection of anti-β2GP1 peptides antibody in vivo. The titers of anti-β2GP1antibodies in the mouse peripheral blood and the activated partial thromboplastin time （APTT） were detected. Results The purity of chemically synthesized β2GP1 peptides was 94%, which met the immunogen standard. The titer of antiserum of the rabbit immunized with β2GPI peptide coupling with KLH was over 1：32 000. Western blotting showed that the anti-β2GP1 peptides antibody could specifically recognize both human and mouse β2GP1. Furthermore, ELISA showed that the antibody could specifically bind to β2GP1 cryptic epitope. In vitro experiments demonstrated that the anti-β2GP1 peptides antibody/ β2GP1 complexes could enhance p38 and NF-KB p65 phosphorylation in mouse peritoneal macrophages and induce TF mRNA and protein expression. Moreover, EAPS mouse model induced by anti-β2GP1 peptide antibody was successfully established, in which the titers of anti-β2GP1 antibody in mouse peripheral blood were greater than 1：3200 and APTT was significantly shorter that that of control group. Conclusion The anti-β2GP1 peptide antibody we prepared could specifically recognize both human and mouse β2GP1 and specifically bind to β2GP1 cryptic epitope. It was also proved to have the pathogenic effect.