棉花咖啡酸-O-甲基转移酶基因的原核表达及蛋白纯化鉴定  

Prokaryotic Expression,Protein Purification and Western Blotting Identification of GhCOMT2 Gene from Gossypium hirsutum L.

作　　者：李波[1,2] 倪志勇[1] 石庆华[2] 范玲 

机构地区：[1]新疆农业科学院,核技术生物技术研究所,乌鲁木齐830091 [2]新疆农业大学农学院,乌鲁木齐830052

出　　处：《西北植物学报》2010年第9期1738-1743,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家“863”项目(2006AA10Z184);农业部转基因重大专项(2009ZX08005-011B);新疆自治区高技术研究发展计划项目(200611101);国家自然科学基金(30660088)

摘　　要：为获得大量高纯度的GhCOMT2蛋白以便研究其功能和性质,以pMD18-GhCOMT2质粒为模板,PCR扩增GhCOMT2基因的cDNA编码区,构建原核表达载体pET-28a-GhCOMT2,经酶切鉴定并测序后转化到大肠杆菌BL21(DE3)中进行诱导表达,并采用Western blotting方法鉴定表达产物。结果表明:在大肠杆菌BL21(DE3)菌株中成功表达了与标签蛋白融合的GhCOMT2蛋白,大小约为40.062 kD,浓度为0.62 mg/mL。重组蛋白的最佳诱导条件为:0.2 mmol/LIPTG在16℃诱导12 h。重组蛋白以可溶形式高效表达,用蛋白标签亲和层析柱(HisTrapTMHP)获得纯化重组蛋白,Western blotting分析表明其能与His多克隆抗体起特异性反应。To obtain the GhCOMT2 protein with high purity in a large scale and investigate the function of GhCOMT,the cDNA of GhCOMT2 gene was amplified by PCR using pMD18-GhCOMT2 as template and the full-length open reading frame was fused into the prokaryotic expression vector pET-28a-GhCOMT2.Double digestion with endonucleases showed that the recombinant vector pET-28a-GhCOMT2 was accurately constructed and transformed into E.coli BL21（DE3） cells.Then Western blotting was employed to identify the target protein.The results indicated that the pET-28a-GhCOMT2 with the predicted molecular weight of about 40.062 kD was successfully expressed in E.coli BL21（DE3） strain with the optimal condition by 0.2 mmol/L IPTG treatment for 12 h at 16℃.The concentration of purified protein was 0.62 mg/mL.The fusion protein mainly appeared as dissoluble form.Western blotting indicated that the fusion protein purified by His TrapTM HP could be combined with antibody effectively as expected.All these results suggested that GhCOMT2 protein was highly expressed in E.coli.

关 键 词：GhCOMT2 原核表达 蛋白纯化 WESTERN BLOTTING 

分 类 号：Q786[生物学—分子生物学] S562[农业科学—作物学]