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2025年8月11日 星期一
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基于途径工程的极大螺旋藻藻蓝蛋白α亚基的生物合成
于平
浙江工商大学
The Biosynthesis of the Phycocyanin Holo-α-subunit from Spirulina maxima Based on Pathway Engineering
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摘要 本文根据极大螺旋藻藻蓝蛋白α亚基的生物合成途径,利用PCR技术从极大螺旋藻基因组DNA中克隆了与其生物合成相关的五个基因cpcA、hox1、pcyA、cpcE和cpcF,并将这些基因用相应的限制性内切酶酶切后,依次构建到表达载体pETDuet-1的两个外源基因表达盒内,转化大肠杆菌BL21 (DE3),经氨苄青霉素抗性筛选,得到工程菌ZJGSU02。经测序确认,连接到pETDuet-1上的五个基因拼接正确。IPTG诱导后,工程菌培养液中的细胞呈现明显的蓝色,对照菌颜色没有发生变化。SDS-PAGE胶显示在21kDa处有一明显的条带,与预期理论分子量大小一致。重组蛋白经锌离子溶液浸泡后,在紫外光激发下呈现桔色荧光,western blotting分析表明重组蛋白能特异性地与6×His-tag单克隆抗体结合。通过吸收光谱和荧光光谱检测,发现重组蛋白最大吸收波长为623nm,最大荧光发射波长为645.8nm,与天然极大螺旋藻中藻蓝蛋白α亚基的最大吸收波长和最大荧光发射波长一致,以上结果表明极大螺旋藻藻蓝蛋白α亚基已在大肠杆菌中成功实现异源表达。该研究结果不仅为色基结合蛋白这类复杂蛋白在异源宿主系统中的表达提供新的思路和方法,而且也为探索在一个表达载体上构建和表达5个或5个以上的外源目的蛋白基因及其基因互作等方面的研究提供借鉴,具有重要意义。
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于平
关键词 极大螺旋藻 藻蓝蛋白 异源表达 途径工程    
Abstract:Five genes (cpcA, hox1, pcyA, cpcE and cpcF), involved in the biosynthesis of phycocyanin holo-α-subunit, were cloned from the genomic DNA from Spirulina maxima. These genes were digested by the corresponding restriction enzymes and sequentially ligated into the expression plasmid pETDuet-1 with two multiple cloning sites to construct the expression vector pETDuet-6. The recombinant vector pETDuet-6 was then transformed into E. coli BL21 (DE3) and ampicillin resistant transformant ZJGSU02 was selected. The fidelity of the recombinant vector was confirmed by DNA sequencing. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG), the cells in the culture of the engineered strain exhibited a pronounced blue. The color change was not seen in the cells in the culture of the control strain. Visualization of the proteins on coomassie-stained SDS-PAGE gel showed about 21kDa distinct band. After the SDS-PAGE gel being soaked in zinc ion solution, a bright fluorescence band was visualized by UV illumination. Western blotting indicated the specific binding of the recombinant protein to 6×His-tag monoclonal antibody. The absorption spectrum of the recombinant phycocyanin holo-α-subunit had a λmax at 623nm, and the maximum fluorescence emission wavelength of it was at 645.8nm, which were in agreement with those of the natural phycocyanin holo-α-subunit from S. maxima. These results demonstrated that phycocyanin holo-α-subunit of S. maxima was successfully expressed in the heterologous E. coli. This study provided not only a new strategy for the expression of pigment-binding protein in the heterologous host, but also a reference for exploring the construction and expression of five or more heterologous protein genes using one vector and the study of the interaction among the genes.
Key wordsSpirulina maxima    phycocyanin    heterologous expression    pathway engineering
收稿日期: 2009-03-24     
通讯作者: 于平   
引用本文:   
于平. 基于途径工程的极大螺旋藻藻蓝蛋白α亚基的生物合成[J]. , 2010, 18(3): 501-507.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2010/V18/I3/501
 
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