Sadeghi H Mir Mohammad, Ahmadi R, Aghaabdollahian S, Mofid M R, Ghaemi Y, Abedi D
Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Science, Isfahan, I.R.Iran.
Res Pharm Sci. 2011 Jan;6(1):51-5.
Due to the widespread applications of xylitol dehydrogenase, an enzyme used for the production of xylitol, the present study was designed for the cloning of xylitol dehydrogenase gene from Glcunobacter oxydans DSM 2003. After extraction of genomic DNA from this bacterium, xylitol dehydrogenase gene was replicated using polymerase chain reaction (PCR). The amplified product was entered into pTZ57R cloning vector by T/A cloning method and transformation was performed by heat shocking of the E. coli XL1-blue competent cells. Following plasmid preparation, the cloned gene was digested out and ligated into the expression vector pET-22b(+). Electrophoresis of PCR product showed a 789 bp band. Recombinant plasmid (rpTZ57R) was then constructed. This plasmid was double digested with XhoI and EcoRI resulting in 800 bp and 2900 bp bands. The obtained insert was ligated into pET-22b(+) vector and its orientation was confirmed with XhoI and BamHI restriction enzymes. In conclusion, in the present study the recombinant expression vector containing xylitol dehydrogenase gene has been constructed and can be used for the production of this enzyme in high quantities.
由于木糖醇脱氢酶（一种用于生产木糖醇的酶）的广泛应用，本研究旨在从氧化葡萄糖杆菌DSM 2003中克隆木糖醇脱氢酶基因。从该细菌中提取基因组DNA后，使用聚合酶链反应（PCR）复制木糖醇脱氢酶基因。通过T/A克隆方法将扩增产物导入pTZ57R克隆载体，并通过热激处理大肠杆菌XL1-blue感受态细胞进行转化。质粒制备后，将克隆的基因切出并连接到表达载体pET-22b(+)中。PCR产物的电泳显示出一条789 bp的条带。然后构建了重组质粒（rpTZ57R）。该质粒用XhoI和EcoRI进行双酶切，产生800 bp和2900 bp的条带。将获得的插入片段连接到pET-22b(+)载体中，并用XhoI和BamHI限制性酶确认其方向。总之，在本研究中构建了含有木糖醇脱氢酶基因的重组表达载体，可用于大量生产该酶。
