基于人脂多糖结合蛋白和聚羟基脂肪酸结合蛋白PhaP的内毒素去除方法

发布时间：2018-03-24 05:45

  本文选题：生物制品　切入点：聚羟基脂肪酸　出处：《汕头大学》2011年硕士论文

【摘要】：内毒素是革兰氏阴性菌细胞壁外壁中的特有结构,是其在生长、分裂或死亡过程中释放到环境中的有毒物质。内毒素分子化学性质非常稳定,并且在水溶液中易聚合成不同分子量的聚合物,使得生物制品中内毒素的去除变得非常困难。聚羟基脂肪酸酯(PHA)颗粒结合蛋白质(PhaP)对PHA以及其它疏水性聚酯具有较强的亲和力。人脂多糖结合蛋白(hLBP)是人体血浆内自然存在的内毒素受体。在本论文研究中,融合蛋白rhLBP PhaP基因在毕赤酵母GS115中分泌表达。收集到的融合蛋白通过PhaP锚定在PHB颗粒表面,用于水和蛋白质溶液中内毒素的去除研究。牛血清白蛋白(BSA)溶液,卵清蛋白(ovalbumin)溶液,胰凝乳蛋白酶原(basic proteinα-chymotrypsinogen)溶液三种蛋白质溶液作为模式被使用,用于研究蛋白质溶液中内毒素的去除效率和蛋白质的回收率,以及离子强度和酸碱度对去除效果的影响。结果表明:本论文研究制作的亲和颗粒(rhLBP- PhaP-PHB颗粒)的内毒素去除率可达90%以上,并且内毒素的去除率和蛋白质的回收率几乎不受离子强度的影响,但是酸碱度对其有一定的影响。我们的研究结果表明,基于rhLBP- PhaP-PHB颗粒的内毒素去除系统,具有准备简单,无毒和高效的特点,非常适合蛋白质纯化工艺中内毒素的去除。
[Abstract]:Endotoxin is a unique structure in the outer wall of Gram-negative bacteria. It is a toxic substance released into the environment during growth, division or death. The molecular chemical properties of endotoxin are very stable. And it is easy to polymerize into polymers with different molecular weights in aqueous solutions. It is very difficult to remove endotoxin from biological products. PhaP) has strong affinity to PHA and other hydrophobic polyester. Human lipopolysaccharide binding protein hLBPis human plasma. Endogenous endotoxin receptor. The fusion protein rhLBP PhaP was secreted and expressed in Pichia pastoris GS115. The fusion protein was anchored on the surface of PHB particles by PhaP for the study of endotoxin removal in water and protein solution. Three protein solutions of chymotrypsin solution, basic protein 伪 -chymotrypsinogen solution, were used as models to study the removal efficiency of endotoxin in protein solution and the recovery rate of protein. The effect of ionic strength and pH on the removal of endotoxin was also studied. The results showed that the endotoxin removal rate of the affinity particle (rhLBP- PhaP-PHB) was over 90%. The removal rate of endotoxin and the recovery rate of protein are almost independent of ionic strength, but the pH has a certain effect on it. Our results show that the endotoxin removal system based on rhLBP- PhaP-PHB particles has a simple preparation. The characteristics of non-toxic and high efficiency are very suitable for removing endotoxin in protein purification process.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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