枯草芽孢杆菌bioI基因的克隆和高效表达及BioI蛋白的纯化  

Cloning and high-level expression of Bacillus subtilis gene bioI and purification of protein BioI

作　　者：龚月生[1] 王晶[1,2] 刘锦妮[1,2] 袁新宇[2,3] 姬生跃[1,2] 王俊 杨明明[1,2] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]武汉阳光广济医药开发有限公司,湖北武汉430064 [3]湖北工业大学生物工程学院,湖北武汉430064

出　　处：《西北农林科技大学学报（自然科学版）》2008年第12期35-40,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家重点新产品计划项目(2003ED760039)

摘　　要：【目的】克隆和表达枯草芽孢杆菌(Bacillus subtilis)bioI基因并纯化表达产物。【方法】提取B.sub-tilis1A747基因组DNA,从中扩增bioI基因并将其克隆到大肠杆菌(Escherichia coli)的表达载体pET-28a(+)上,构建bioI的表达载体pET28a-bioI。将重组质粒pET28a-bioI电转化到大肠杆菌表达宿主BL21(DE3)中进行IPTG诱导表达,对表达产物BioI用Ni2+-NAT亲和层析树脂进行纯化,并对纯化蛋白进行波长扫描。【结果】成功构建了表达载体pET28a-bioI,酶切鉴定结果正确并在大肠杆菌中表达成功,经IPTG诱导8 h后,重组蛋白BioI的表达量占总可溶性蛋白的20%;波长扫描发现,重组蛋白BioI在400 nm处有特征吸收峰。【结论】bioI基因克隆表达成功,其表达产物BioI具有P450蛋白的特征,为蛋白性质的进一步研究和抗体的制备奠定了基础。[Objective] The study was done to clone and express bioI in Escherichia coli and purify the expression protein. [Method] The genome DNA was extracted and gene bioI was amplified by polymerase chain reaction （PCR） from the Bacillus subtilis and cloned into the expression vector pET-28a （＋）,yield- ing recombinant pET28a-bioI. For overproduction of the bioI, gene bioI was successfully expressed and the protein BioI was purified via the Ni-NAT resin. Moreover, the recombined BioI was scanned by different spectra waves. [Result] Expression vector was constructed successfully and gene bioI was successfully expressed in E. coli BL21 （DE3）after adding Isopropyl-β-D-thiogalactopyranoside （IPTG） for 8 hours. The expression product protein BioI made up 20% of total soluble protein through SDS-PAGt~ analysis. It showed a characteristic absorption peak in 400 nm by different spectra waves. [Conclusion] The gene bioI was successfully expressed in E. coli and it had the character of P450 protein. These laid a foundation for the research of the protein character and antibody preparation.

关 键 词：枯草芽孢杆菌 bioI基因 大肠杆菌BL21(DE3) 高效表达 

分 类 号：Q786[生物学—分子生物学]