自制肝素亲和柱分离纯化载脂蛋白H及其抗体制备与鉴定  

Isolation and purification of apolipoprotein H with heparin affinity column and preparation and identification of anti-ApoH

作　　者：王相栋[1] 张春妮[1] 汪俊军[1] 田迎[1] 

机构地区：[1]南京大学医学院临床学院南京军区南京总医院检验科,南京210002

出　　处：《中国免疫学杂志》2008年第10期931-934,942,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(No30471649);江苏省"六大人才高峰"基金(No20050203)

摘　　要：目的:自制肝素-琼脂糖6B亲和柱提纯人血清载脂蛋白H(apoH),免疫制备多克隆抗体,为大量制备与进一步研究做准备。方法:利用间接还原胺化法制备亲和层析柱,并优化反应条件。运用高氯酸沉淀、离子交换及亲和层析从人血清中纯化抗原,制备兔源多克隆抗体。结果:自制亲和柱肝素结合量约5mg/g(介质体积)。纯化的抗原经SDS-PAGE鉴定分子量约50kD,无杂带;16种氨基酸组成分析结果与报道一致。多克隆抗体与购置的国外抗体一样,在进行ELISA与dot-ELISA时与小牛血清白蛋白有交叉反应;但在变性且还原的条件下,Western blot结果显示无交叉反应。结论:自制亲和柱成本低、亲和性好、肝素结合稳定,用之分离纯化得到了高纯度apoH;兔多克隆抗体用于检测血清中载脂蛋白H含量时需进一步纯化。Objective：To purify apolipoprotein H （apoH） from fresh human serum with heparin affinity column self-prepared and to prepare polyclonal antibody anti-ApoH.Methods：Affinity column was made by reaction of indirect reductive amination and operating conditions were optimized and their effects were investigated.The apolipoprotein H antigen was purified from human serum by methods of perchloric acid precipitation,ion exchange and affinity chromatography.The polyclonal antiserum against the antigen was obtained by immuning rabbits.Results：The amount of heparin immobilized on the synthesized gel was 5 mg/ml （medium）.Molecular weight of the purified antigen was about 50 kD by SDS-PAGE and no other bands were seen,the percentages of 16 amino acids were consistent with that reported previously.In ELISA and dot-ELISA,the polyclonal antibody displayed a cross reaction with bovine serum albumin,while no such reaction was observed in result of Western blot under reduced and denatured condition.Conclusion：The self-prepared affinity column costs very low,but has excellent affinity performance and stable heparin linking,and high purified apoH can be obtained by using the reagent.Polyclonal antibody needs to be further purified before detecting serum apolipoprotein H.

关 键 词：亲和层析 肝素 载脂蛋白H 抗体 纯化与鉴定 

分 类 号：R392-33[医药卫生—免疫学]