【摘要】 [目的]利用哺乳细胞表达系统表达人ATX蛋白,并对其进行纯化、酶学性质分析。[方法]以pFastBacTMHTAATX质粒为模板,PCR扩增ATX基因,并连接至质粒pInsulator-8His,构建真核表达质粒pInsulator-ATX-8His,经双酶切鉴定后,并瞬时转染至HEK293F细胞,SDS-PAGE检测ATX的表达。通过Ni柱和凝胶层析两步法纯化ATX,HPLC鉴定ATX蛋白的纯度。利用Bis-pNPP底物检测纯化ATX的活性及酶学性质。[结果]真核表达质粒pInsulator-ATX-8His构建成功,并在HEK293F细胞中成功表达,经纯化后ATX蛋白纯度达到98%,并且纯化的ATX具有磷酸二酯酶的活性。酶学性质分析,该酶反应最适pH值为9.0,在0⁵0℃有较好的热稳定性,金属螯合剂EDTA可抑制ATX活性,金属离子Ca²⁺、Mg²⁺、Zn²⁺促进ATX的活性。[结论]实现了人ATX在哺乳细胞中的表达,获得了高纯度的ATX蛋白,完成了ATX酶学性质的分析。更多还原

【Abstract】 [Objective]To take advantage of a mammalian expression system to express,purify human autotaxin,and analyzed for ATX enzymatic properties. [Methods] With pFastBacTMHTA-ATX plasmid as a template,autotaxin gene fragment was amplified by polymerase chain polymerization reaction（ PCR）,and inserting into pInsulator-8 His vector. The recombinant pInsulator-ATX-8 His was constructed. After double digestion,the recombinant plasmid was transiently transfected into HEK293 F cells. The expressed autotaxin protein was analyzed by SDS-PAGE. The protein was purified from cells conditioned medium by affinity chromatography with Ni sepharose and gel column chromatography. the purity of protein was determined by HPLC. The enzyme activity of autotaxin was analyzed by Bis-pNPP substrate. [Result]The pInsulator-ATX-8 His was successfully constructed. The autotaxin was successfully expressed in HEK293 F cells. The protein purity quotient was about 98% after purification. ATX protein had phosphodiesterase activity. The enzymatic properties were analyzed. The optimal pH of autotaxin were 9. 0. The purified enzyme had better stability over a temperature range form 0 50 ℃. In addition,the ATX is strongly inhibited by EDTA. The metal ions including Zn²⁺,Mg²⁺and Ca²⁺activated the catalytic activity. [Conclusion] The autotaxin was successfully expressed in mammalian cells and purified to obtain high purity ATX protein,and completed the analysis of enzymatic properties.更多还原