利用气相二氧化硅从人血浆中制备载脂蛋白A1方法的建立

Development of a method for preparation of apolipoprotein A1 from human plasma by fumed silicon dioxide

目的建立利用气相二氧化硅从人血浆中制备载脂蛋白A1(apolipoprotein A1,apo A1)的方法,并对该方法的效果进行评价。方法在人血浆中加入气相二氧化硅吸附apo A1,考察气相二氧化硅加入人血浆比例(m/V)及孵育时间对吸附的影响,确定最佳吸附条件。吸附后,离心分离人血浆与气相二氧化硅,再用不同浓度的乙醇溶液洗脱气相二氧化硅吸附的apo A1,确定乙醇溶液的最佳洗脱浓度。洗脱产物采用BCA法测定总蛋白浓度后,经SDS-PAGE,采用凝胶成像分析软件Bandscan 5.0分析apo A1纯度,并评价该方法制备apo A1的效率。结果人血浆中加入1%(m/V)的气相二氧化硅时,apoA1提取效果最佳。气相二氧化硅与人血浆混合物孵育10 min,可完成对apoA1的饱和吸附。该混合物固液分离在大于12 000×g的离心力条件下离心10 min,可获得最佳效果。洗脱液中乙醇最佳浓度为20%(V/V)。在该洗脱条件下,可从1 mL人血浆中制备纯度为78.7%的apoA1 0.92 mg。结论建立了利用气相二氧化硅从人血浆中制备apoA1的方法,该方法可简便高效地从人血浆中制备apoA1。

Objective To prepare apolipoprotein A1 （apoA1） from human plasma by using fumed silicon dioxide and to evaluate the effect of this method. Methods Fumed silicon dioxide was added into human plasma to adsorb apoA1, and the effects of proportion （m / V ） of fumed silicon dioxide added into human plasma and incubation time on the adsorption efficiency were investigated to optimize the adsorption condition. After adsorption, the fumed silicon dioxide was separated from the plasma by centrifugation, on which the adsorbed apoA1 was washed by alcohol solution at various concen- trations, and the alcohol concentration in the elution solution was optimized. The eluted product was determined for protein concentration by BCA method, from which the proteins were separated by SDS-PAGE. The purity of apoA1 was analyzed by gel imaging analysis software to evaluate the efficiency of this method. Results Addition of 1% （m/V ） of fumed silicon dioxide into human plasma was optimal to extraction of apoA1. The saturated adsorption of apoA1 was completed by in~：ubation of the mixture of fumed silicon dioxide and human plasma for 10 min. The centrifugation at a centrifugal lbree of more than 12 000 x g for 10 min was optimal for the solid-liquid separation of the mixture. The optimal concentration of ethanol in the elution buffer was 20% （V/V） and, under this condition, 0. 92 mg of apoA1 with a purity of 79% was prepared from 1 mL of human plasma. Conclusion A method for preparation of apoA1 from human plasma by fumed silicon dioxide was developed, which was simple and effective.