摘要
人们新近研究发现─种血浆糖蛋白β_2glycoproteinI(β_2GPI)参与抗磷脂抗体(ACL)与心磷脂(CL)的结合反应,要深入研究β_2GPI与ACL、CL三者的关系和ACL在血栓形成发病机理中的作用,首先需将其进行分离、纯化、鉴定。用高氯酸沉淀正常人血清弃杂蛋白,再经过DEAE纤维素离子交换,SephadexG-75凝胶过滤和ProteinA-Sepharose,Heparin-SepharoseCL-6B二步亲和层析分离纯化了β_2GPI,用SDS-PAGE电泳鉴定其分子量为50kD,凝胶扫描其纯度为93.44%,等电点6.1~6.5,氨基酸组成比例分析纯化样品与标准品β_2GPI相符。进─步将纯化鉴定的样品β_2GPI与佐剂混匀,免疫新西兰兔,成功地制备了抗β_2GPI抗体,用对流免疫电泳鉴定制备的抗体可与纯化的样品、β_2GPI标准品形成反应沉淀线,制备的抗体效价大于1∶32。研究意义在于探讨用免疫生化的方法分离心磷脂,纯化了β_2GPI,成功地制备了抗β_2GPI抗体,为深入研究β_2GPI、ACL在血栓形成发病机理中的作用创造了良好的工作基础。
Antiphospholipid antibodies was a kind of important antibodies, which was associated with thrombosis , fetal loss or thrombocytopenia.A plasma lipoprotein had recently been found and identified as β_2-glycoprotein Ⅰ(β_2 GPI)which was required for anticardiolipin antibodies to bind cardiolipin,The β_2 GPI was isolated from plasma,purified by gel filtration,ion exchange chromatography and heparin affinity chromatography.Its molecular weight(50kD)was determinted using SDS-Polyacrylamide electrophoresis.Gel scaning indicated that purity of β_2 GPI was 93 percent. The composition of β_2 GPI amino acid is analysed and was similar to standard β_2 GPI.The β2 GPI migrates at pH 6.1~6.5 on isoelectrofocusing electrophoresis.Prepartion of antisera was obtained by immunized rabbits withβ_2 GPI.The antisera may react with β_2 GPI and produce precipitable band,The titer of antisera was about 1∶32 using immune doubled diffusition,The experimental result was useful to learn about role of β_2 GPI in the pathogenesis of antiphospholipid antibodies syndrome.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
1996年第5期303-306,共4页
Chinese Journal of Immunology