| 摘要: |
| 【目的】构建一种可获得Sumo修饰蛋白的原核表达系统，为Sumo化修饰蛋白的批量制备奠定基础。【方法】用Trizol裂解法提取小鼠畸胎瘤细胞F9的总RNA，反转录成cDNA后PCR扩增获得Sumo1、Ubc9、Sae2和Sae1基因。将上述基因及相应载体酶切、连接后构建出重组质粒pACYC-Ubc9-Sumo1、pCDF-Sae2-Sae1。采用质粒转化制备阳性克隆感受态细胞质粒转化的方法获得pACYC-Ubc9-Sumo1和pCDF-Sae2-Sae1共表达的Sumo化修饰的原核表达系统。以pGEX-GST-Sox2和pGEX-GST-Sox2K247R重组质粒为例，采用Western Blot检测蛋白质Sumo化修饰原核表达系统的有效性。用另一Sumo修饰底物Oct4及其Sumo修饰位点突变的突变体Oct4K118R转化原核Sumo修饰系统，并连续传代，检测蛋白质Sumo化修饰系统的实用性及遗传稳定性。【结果】构建的蛋白质Sumo化修饰原核表达系统可特异修饰Sumo1的底物Sox2和Oct4；含有Oct4原核表达载体的蛋白质Sumo化修饰系统可稳定传至15代，系统的稳定性较好。【结论】获得了成本较低、特异性强、重复性好、稳定性强的可用于制备Sumo化蛋白的原核表达系统。 |
| 关键词: Sumo 蛋白质体外修饰 原核表达 Oct4 Sox2 |
| DOI： |
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| 基金项目:国家自然科学基金项目(30870266) |
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| Construction of protein Sumoylation system in prokaryotic cell |
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| Abstract: |
| 【Objective】The protein Sumoylation system was constructed in Prokaryotic cell to lay a foundation for quantity production of sumoylated protein.【Method】Total RNA was extracted from the mouse teratoma cells F9 with Trizol reagent,reverse transcripted to cDNA,thus obtaining the genes of Sumo1,Ubc9,Sae2,Sae1 fragments by the standard PCR from cDNA.The above fragments and vectors were double digested with enzymes respectively,then they were ligated to constructed recombinate plasmids.The Sumo ylation prokaryotic expression system of pACYC-Ubc9-Sumo1 and pCDF-Sae2-Sae1 co-expressioned was constructed by plasmid transfection,preparing the competent cell of positive clone and plasmid transfection methods.Taking pGEX-GST-Sox2 and pGEX-GST-Sox2K247R for examples,the effectiveness of the protein Sumo ylation system was detected by Western Blot.Another Sumo substrate Oct4 and its mutant Oct4K118R were transfected to Sumo ylation system and the system was passed contiously to measure the practical applicability and stability of the system.【Result】Wild type Sox2 and Oct4,not their mutants,were specially Sumo ylated by the Sumo ylation system.The protein Sumo ylation system with pGEX-GST-Oct4 was stable at least to 15 times subculture.【Conclusion】The system which we constructed is stable,and characterized with lower cost,strong specificity,good repeatability and can be used to get the Sumo ylated proteins. |
| Key words: Sumo in vitro protein modification prokaryotic expression Oct4 Sox2 |
