梯度pH共沉淀法回收人工肝丢弃血浆蛋白初步实验  

Gradient pH co-precipitation to recover proteins from plasma discarded in artificial-liver treatment

作　　者：徐兵[1] 潘晨[1] 蒋晓织[1] 林曼芝[1] 陈敏[1] 林灼[1] 陈怡[1] 陈惠聪[1] 

机构地区：[1]福州市传染病医院肝病研究所,350025

出　　处：《中华传染病杂志》2007年第5期268-271,共4页Chinese Journal of Infectious Diseases

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。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% （29 g/57 g） of the total plasma proteins had been recovered by the gradient pH salting out and 41% （25 g/61 g） by the gradient pH low-temperature ethanol co-precipitation. 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）. Conclusions The proteins with close isoelectric point can co-precipitated with the presence of high concentration 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.

关 键 词：肝 人工 氢离子浓度 沉淀 盐析法 血蛋白质类 回收 

分 类 号：R459.5[医药卫生—治疗学]