【摘要】 目的建立在体外快速回收人工肝丢弃血浆总蛋白的方法。方法设计一种梯度pH共沉淀法,从pH 7.3开始,逐滴加入1 mol/L柠檬酸溶液,5h加完,使溶液pH值呈渐进梯度变化,至终点pH 5.1,用此法配合盐析法(氯化钠饱和度33%,4℃反应5.5h)或低温乙醇沉淀法(乙醇体积分数为0.04,-7℃反应5.5h),使等电点在pH 7.3～5.1范围内的各蛋白连环碰撞聚集而析出,形成多蛋白共沉淀,再一并回收,弃上清液以清除毒物。结果在pH 7.3～5.1范围内,用梯度pH低温乙醇法和梯度pH盐析法分别回收了41%(25 g/61 g)和50%(29 g/57 g)的血浆蛋白,上清液中未回收的蛋白主要是白蛋白及其所结合的胆红素。两法回收的蛋白溶液中,TBil下降,分别为回收前的0.07%(低温乙醇法)和0.06%(盐析法),HBV DNA降为阴性(PCR)。结论在高浓度中性盐或低温乙醇条件下,渐进微调溶液pH值,可使一连串有相邻等电点的蛋白共沉淀,采用梯度pH共沉淀法可选择性快速回收人工肝丢弃血浆中总蛋白。更多还原

【Abstract】 Objective To set up a system in vitro to rapidly recover plasma proteins lost during artificial-liver treatment.Methods The polyprotein precipitation was obtained by all proteins whose isoelectric point pH value were between 7.3 and 5.1,which collided with each other and aggregated using gradient pH co-precipitation(adding 1 mol/L citric acid slowly in the plasma solution to change the pH values gradually from 7.3 to 5.1 in 5 h)combined with salting out(degree of saturation of NaCl is 33%,reacted for 5.5 h at 4℃)or low-temperature ethanol precipitation(40% ethanol, reacted for 5.5 h at -7℃)so that to get rid of toxicants by discarding the supernatant.Results In the range of pH 5.1-7.3,50%(29g/57g)of the total plasma proteins had been recovered by the gradient pH salting out and 41%(25 g/61g)by the gradient pH low-temperature ethanol co-precipi- tation.The protein remained in the supernatant was mostly albumin and its combined bilirubin.The levels of total bilirubin decreased to 0.07% and 0.06% of the original levels by these two methods respectively and the serum HBV DNA level decreased to be undetected(quantitative PCR).Conclu- sions The proteins with close isoelectric point can co-precipitated with the presence of high concen- tration of NaCl or low-temperature ethanol and by changing the pH value gradually.The total protein in the discarded plasma during artificial-liver treatment can be recovered rapidly using the gradient pH coprecipitation.更多还原