GA2ox1氧化酶基因的克隆及原核表达  

Cloning and Prokaryotic Expression of GA2ox1 Gene

作　　者：常丽[1] 赵小英[1] 郭明[1] 林建中[1] 李秀山[1] 刘选明[1] 

机构地区：[1]湖南大学生命科学与技术研究院,长沙410082

出　　处：《西北植物学报》2010年第6期1099-1104,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家863项目(20070349669);国家自然科学基金(30800080;30770200)

摘　　要：采用RT-PCR从拟南芥基因组中克隆到GA2ox1基因,通过Gateway克隆技术构建原核重组表达载体pD-EST17-GA2ox1,并转化大肠杆菌BL(21)star,对蛋白原核表达条件进行优化。结果表明,最佳表达条件为温度30℃、IPTG浓度0.2 mmol/L、诱导6 h。蛋白表达形式为包涵体,与Biotech对GA2ox1基因在大肠杆菌中的表达情况推测的结论一致。重组蛋白分子质量约为40 kD,经Ni Sepharose亲和层析柱纯化和Western blot鉴定得纯度90%以上的GA2ox1重组蛋白。研究结果为GA2ox1抗体制备及蛋白功能的进一步研究奠定了基础。GA2ox1 gene was cloned by RT-PCR from Arabidopsis thaliana genome.Prokaryotic expression vector pDEST17-GA2ox1 was constructed by Gateway Clone Technology,and transformed into E.coli BL（21） star for expression,then we determined the optimal expression condition.Our results showed the optimal condition for prokaryotic expression was that induction could be performed at 30℃ for 6 h with 0.2 mmol/L IPTG.GA2ox1 gene was successfully expressed in E.coli BL（21）star in the form of inclusion bodies,which was consistent with the speculation of Biotech.90 percent of a 40 kD recombinant protein of GA2ox1 was purified through Ni Sepharose Purification System and detected by Western Blot.This study establishes a basis for antibody preparation and further functional studying of GA2ox1.

关 键 词：GA2ox1基因 Gateway克隆 原核表达 纯化 WESTERN BLOT 

分 类 号：Q786[生物学—分子生物学]