| 摘要: |
| [目的]为人类泛素样小蛋白4(SUM04)多克隆抗体制备和疾病研究奠定基础.[方法]根据Gen-Bank提供的核酸序列,设计7条单链DNA,采用重叠延伸PCR方法,合成SUMO4基因编码区.基因片段经限制性内切酶双酶切,构建到表达载体pGEX-4T-1中.酶切、测序鉴定后,将重组子转染BL21 RIL菌株,表达及纯化融合蛋白.用SDS-PAGE和Western blot检测纯化效果,鉴定表达产物.[结果]成功合成了长度约为280 bp的SUMO4基因,经双酶切鉴定,SUMO4原核表达载体构建成功,插入片段测序正确.GST-SUMO4融合蛋白在终浓度1mmol/L IPTG、28℃诱导5 h后产量达到高峰,SDS-PAGE证实表达产物以可溶形式存在,分子量约为37 ku,GST亲和层析的纯化效果良好,Western blot验证融合蛋白分子量与预期值相符.[结论]SUMO4蛋白在大肠杆菌中高效表达,并以可溶性蛋白形式存在. |
| 关键词: 人类泛素SUMO4 重叠延伸PCR 原核表达 蛋白纯化 |
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| Cloning, prokaryotic expression, purification and identification of human small ubiquitin-related modifier 4 (SUM04) |
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| Abstract: |
| 【Objective】 Homo－SUMO4 was artigicially synthesized and expressed to provide a basis of related medical research and preparation of polyclonal antibody against SUMO4. 【Method】 Based on the DNA sequence from GenBank,seven single－strand DNA were designed for amplification of SUMO4 through SOE PCR（gene splicing by overlap extension PCR）.The fragment was digested with restriction endonucleases,recombined into pGEX－4T－1.After restricting enzyme identification and sequencing,expression of fusion protein was induced after transformation into the E.coli strain BL21 RIL,followed by purification through affinity chromatography.SDS-PAGE and Western blot were then employed to identify target protein. 【Result】 SUMO4 prokaryotic expression vector was successfully constructed with correct sequence.The molecular mass and solubility of GST-SUMO4 were proved by SDS-PAGE and Western blot.The purification effect was good.【Conclusion】 SUMO4 could be highly expressed in E.coli as soluble protein. |
| Key words: SUMO4 SOE PCR prokaryotic expression protein purification |
