【摘要】 目的分离纯化载脂蛋白M阳性和阴性高密度脂蛋白(ApoM~+ HDL和poM~- HDL),检测其与清道夫受体B类Ⅰ型(SR-BI)的结合能力。方法采用腹水诱生的方法以重组人ApoM免疫小鼠制备小鼠抗人ApoM单克隆抗体。利用该抗体通过亲和层析的方法分离ApoM~+ HDL和poM~- HDL,采用二喹啉甲酸(BCA)蛋白定量方法对其定量分析。通过免疫共沉淀方法检测ApoM~+ HDL和poM~- HDL与巨噬细胞株THP-1细胞表面表达的SR-BI的相互作用。结果经免疫、融合、筛选,获得4株特异性分泌ApoM单抗的杂交瘤细胞株,采用腹水诱生方案进行ApoM抗体的大量表达,获得浓度为1mg/ml的ApoM单抗,在1∶4000稀释后仍能在25kD处检测出目的蛋白。利用ApoM单抗进行亲和层析,分别收集流出液(poM~- HDL)、洗脱液(ApoM~+ HDL),洗脱液中能检测到ApoM,流出液中未检测到ApoM。经BCA蛋白定量poM~- HDL浓度为3.23mg/ml,ApoM~+ HDL浓度为0.34mg/ml。佛波酯处理THP-1细胞后表面SR-BI表达升高10%左右。免疫共沉淀结果显示,poM~- HDL、ApoM~+ HDL孵育组的总蛋白经SR-BI抗体沉淀的复合物中均能检测到SR-BI和HDL,重组人ApoM孵育组也能检测到SR-BI和ApoM。结论小鼠抗人ApoM单抗具有良好的灵敏度和特异度,poM~- HDL和ApoM~+ HDL两种蛋白成分和重组人ApoM均能与THP-1细胞表面SR-BI结合。更多还原

【Abstract】 Objective To separate and purify the apolipoprotein M positive and negative high density lipoprotein(ApoM~+ HDL and poM~- HDL)from human HDL protein and investigate their binding capacities to scavenger receptor class B type Ⅰ(SR-BI),respectivly.Methods Recombinant human ApoM protein was used as antigen to immunize BALB/c mice and the mAbs against ApoM were prepared by hybridoma technique.ApoM~+ HDL and poM~- HDL were separated from human plasma by affinity chromatography.The proteins were quantified by BCA protein assay kit.The binding capacities between ApoM~+ HDL,poM~- HDL,recombinant ApoM protein and SR-BI were examined by co-immunoprecipitation.Results After immunization,cytomixis and screening,4 hybridoma cell lines secreting high titer of ApoM McAbs were obtained.The best hybridoma to produce large quantities of ApoM McAbs(1 mg/ml)was chosen,which could also detect the target protein until 1∶4000 diluted.Human HDL was purified into two components with ApoM McAbs and HiTrap NHS-activated HP column.After immunoaffinity chromatography the effluent(poM~- HDL)and the eluant(ApoM~+ HDL)concentrations were 3.23 mg/ml and 0.34 mg/ml,respectively.Treated by PMA and ox-LDL,the expression of SR-BI on the surface of THP-1 cells increased about 10%.The results of co-immunoprecipitation showed recombinant human ApoM,poM~- HDL and ApoM~+ HDL particles could interact with SR-BI on the surface of THP-1 cell line.Conclusion The mouse anti-human ApoM monoclonal antibody has both high sensitivity and specificity.poM~- HDL,ApoM~+ HDL and recombinant ApoM protein can interact with SR-BI expressed on the surface of THP-1 cells.更多还原