论文部分内容阅读
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。
OBJECTIVE To isolate and purify apolipoprotein M positive and negative high density lipoprotein (ApoM HDL and poM HDL), and to detect its binding ability with scavenger receptor type B type Ⅰ (SR-BI). Methods Mouse anti-human ApoM monoclonal antibody was prepared by immunization of mice with recombinant human ApoM using ascites-induced method. ApoM ~ + HDL and poM ~ - HDL were separated by affinity chromatography using this antibody, and quantified by quantitative analysis of bicinchoninic acid (BCA) protein. The interaction of ApoM ~ + HDL and poM ~ - HDL with SR-BI expressed on the surface of THP-1 macrophage cell line was detected by co-immunoprecipitation. Results Four hybridoma cell lines secreting ApoM McAbs were obtained by immunization, fusion and screening. The expression of ApoM antibody in ascites induced solution was obtained, and the ApoM McAb at the concentration of 1 mg / ml was obtained at 1: 4000 The target protein can still be detected at 25 kD after dilution. ApoM McAb was used for affinity chromatography. The effluent (poM-HDL) and eluate (ApoM + HDL) were collected respectively. ApoM was detected in the eluate and ApoM was not detected in the effluent. After BCA protein was quantified poM ~ - HDL concentration of 3.23mg / ml, ApoM ~ + HDL concentration of 0.34mg / ml. Phosphatidylserine treatment of THP-1 cells surface SR-BI expression increased by about 10%. The result of co-immunoprecipitation showed that SR-BI and HDL could be detected in all the complexes of poM-HDL and ApoM + HDL by SR-BI antibody precipitation, and SR -BI and ApoM. Conclusion The mouse anti - human ApoM McAb has good sensitivity and specificity. Both poM - HDL and ApoM + HDL protein and recombinant human ApoM can bind SR - BI on the surface of THP - 1 cells.