自身抗体的辅助因子β_2GPI分离纯化、鉴定及抗体的制备

ISOLATION,PURIFICATION AND IDENTIFICATION OF β_2 GPIAND PREPARATION OF ITS ANTISERA

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.