人源CLP的表达、纯化及生化特性研究

目的：为进一步研究CLP（coactosin-likeprotein）与5’-脂氧合酶、肌动蛋白的相互作用机制及功能，开展CLP克隆表达、分离纯化研究,以得到高纯度的CLP，并对其生物化学特性进行分析测定。方法：从人的胎肝cDNA文库中经PCR扩增得到CLP基因，克隆到原核表达载体pGEX-6p-1中并获得高效表达，经过Glutathione Sepharose 4B亲和层析和Su-perdex 75分子筛纯化，得到高纯度的CLP；在此基础上进行SDS-PAGE、动态光散射和分析型超速离心等实验，并进一步分析实验结果。结果：CLP在溶液中主要以单体形式呈现；CLP的摩擦率为1．909，证实该蛋白质具有线性化趋势存在的可能性，且线性化程度较高。结论：实验结果揭示了CLP作为线性化蛋白质的可能性，为进一步搭建CLP和丝状肌动蛋白的作用模型奠定了一定的数据基础。

Expression,Purification and Biochemical Characteristic Analyses of Human Coactosin-like Protein

Objective： To clone, express, purify and acquire CLP（coactosin-like protein） with high purity, and to determine and analyze the biochemical characteristics of CLP, for the further research on the function of CLP and the interaction mechanism among CLP, 5＇-lipoxygenase（5LOX） and actin. Methods： The gene of CLP was PCR amplified from human liver cDNA library and cloned into the prokaryotic expression vector pGEX-6p-1. The recombinant CLP was overex- pressed and purified through affinity chromatography（Glutathione Sepharose 4B） and gel filtration chromatography（Superdex 75）. The acquired CLP with high purity was analyzed by SDS-PAGE, dynamic light scattering and analytical ultracentrifuge to get further results. Results： The results showed that the CLP was mainly monomer in solution. The frictionfactor was 1.909, which verified the potential linearization tendency of CLP and the extent of linearization was relatively high. Conclusion： The results suggested the potential linearization tendency of CLP and established a certain experimental basis for us to constitute an interaction model between CLP and F-actin.