摘要
通过RT-PCR获得UDPGDH cDNA序列,经酶切、连接,以pET-32a为原核表达载体,构建成pET-32a-UDPGDH重组表达载体.经酶切、PCR及DNA测序鉴定后,将阳性质粒转化表达受体菌E.coli BL21(DE3),经IPTG诱导后表达出1个约53 000的蛋白,与推测的UDPGDH编码产物的大小一致.凝胶成像分析表明,最高表达量可占菌体总蛋白的47.1%,表明UDPGDH重组载体在大肠杆菌中获得了稳定的表达.
In this study, UDP-glucose dehydrogenase gene was isolated from ramie （Boehmeria nivea L.） by RT-PCR. and cloned into the plasmid pET-32a. The pET-32a-UDPGDH recombinant plasmid was identified by enzyme digesting, PCR and sequencing.The positive recombinant plasmid was subsequently transformed into expression Escherichia coli BL21 （DE3）pLysS. The expression of the recombinant pET-32a-UDPGDH being induced by IPTG led to the production of 53 000 ploypeptide with UDPGDH enzyme activity. The expression yield was about 47. 1% of total bacterial protein. This showed that UDPGDH is a functional gene which could be stably expressed in E.coli. The successful expression of UDPGDH gene in E.coli provides a support for further studying the biological function of UDPGDH.
出处
《湖南农业大学学报（自然科学版）》
CAS
CSCD
北大核心
2008年第3期256-259,297,共5页
Journal of Hunan Agricultural University(Natural Sciences)
基金
国家自然科学基金项目(30571186)
