Separation of Factor V Leiden molecule, a mutated form of Factor V, from plasma of homozygous patient.
Separation of Factor V Leiden molecule, a mutated form of Factor V, from plasma of homozygous patient.
复制标题
从纯合子患者的血浆中分离因子 V Leiden 分子（因子 V 的突变形式）。
DOI：
10.1007/978-0-387-74911-2_12
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发表时间：
2008
影响因子：
--
通讯作者：
Kang,KyungA
中科院分区：
文献类型：
--
作者：
Rezania,Samim;Kang,KyungA
因子V（FV）是血浆中的凝血剂。FV分子由重链和轻链组成，并且因子V Leiden（FVL）是在重链中的单个氨基酸处突变的FV。FVL患者体内处于危险的高凝状态。目前的FVL诊断是通过DNA分析进行的，这是昂贵和耗时的。我们的团队一直在开发用于FVL诊断的实时、成本有效的免疫光学生物传感器。对于传感器的开发，需要目前不可用的纯FVL。在此，我们尝试了从FVL患者的血浆中纯化FVL。由于血浆中含有许多蛋白质，有些蛋白质与FV在结构上同源，因此必须通过非常特异的方法进行纯化，如免疫亲和层析。然而，不与FV反应的抗体目前不可用。因为突变在重链中，并且FVL的轻链的氨基酸序列与FV的氨基酸序列相同，所以测试针对FV的轻链产生的抗体以纯化FVL。从纯合FVL患者获得血浆。首先，通过柠檬酸钡和聚乙二醇6000预处理血浆，以去除维生素K依赖性蛋白、α球蛋白和其他小于6 kDa分子量的蛋白。该方法的产率为54%。然后使用抗FV轻链抗体固定化的CNBr-Sepharose从患者血浆中进行FVL的免疫亲和纯化，纯化产率为25%。总之，针对FV轻链的抗体能够以14%的总产率从血浆中纯化单点突变形式的FV（FVL）。同样的原理也可用于其它单点突变蛋白的纯化。
Factor V (FV) is a coagulant in plasma. The FV molecule consists of a heavy chain and a light chain, and Factor V Leiden (FVL) is mutated FV at a single amino acid in the heavy chain. FVL patients are in a dangerous hyper-coagulation state in their body. Current FVL diagnosis is done by DNA analysis, which is expensive and time consuming. Our group has been developing a real-time, cost effective immuno-optical biosensor for FVL diagnosis. For the sensor development, pure FVL, which is not currently available, is needed. Here, we have attempted FVL purification from FVL patient’s plasma. Since plasma contains many proteins and some proteins are structurally homologous to FV, the purification must be done by a very specific method, such as immuno-affinity chromatography. However, an antibody that does not react with FV is not currently available. Because the mutation is in the heavy chain and the amino acid sequence of the light chain of FVL is identical to that of FV, antibodies generated against the light chain of FV were tested for purifying FVL. Plasma was obtained from a homozygous FVL patient. First, the plasma was pretreated by barium citrate and polyethylene glycol 6000, to remove the vitamin K-dependent proteins, alpha globulins, and other smaller than 6 kDa molecular weight proteins. The yield in the process was 54%. Immuno-affinity purification of FVL from patient plasma was then performed using an anti-FV light chain antibody immobilized CNBr-Sepharose, and the purification yield was 25%. In summary, the antibody against the light chain of FV was able to purify the single point mutated form of FV (FVL) from plasma with an overall yield of 14%. The same principle can probably be used for purification of the other single point mutated proteins.