| 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-tilis 1A747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。 |
| 关键词: 枯草芽孢杆菌 bioI基因 大肠杆菌BL21（DE3） 高效表达 |
| DOI： |
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| 基金项目:国家重点新产品计划项目(2003ED760039) |
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| Cloning and high-level expression of Bacillus subtilis gene bioI and purification of protein BioI |
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| Abstract: |
| 【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 (+),yielding 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-PAGE 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. |
| Key words: Bacillus subtilis bioI E.coli BL21(DE3) high-level expression |
