Cd1-型亚硝酸盐还原酶脱氮工程菌的构建与表达

目的用基因工程技术将铜绿假单胞菌PAO1中nirS基因定向克隆至表达载体pQE-30上，使nirS基因得到高效表达。方法根据GenBank公布的nirS碱基序列和表达载体pQE-30的多克隆位点设计引物，以铜绿假单胞菌PAO1的基因组DNA为模板，应用PCR技术扩增目的片段nirS；之后经过BamH I和HindⅢ双酶切，定向克隆到pQE-30上，化学转化DH5α，构建含有重组质粒的转化子pQE30-nirS-DH5α；经酶切和测序鉴定，扩增产物的碱基序列与GenBank公布的序列完全吻合，再将重组质粒pQE30-nirS转化表达菌株SG13009构建脱氮基因工程菌pQE30-nirS-SG-13009（PNS）。最后用SDS-PAGE（IPTG浓度：0．05mmol／L；诱导温度：25℃；诱导时间：2～3h）和His-tag in-gel Stain鉴定cd1-型亚硝酸盐还原酶的分子量与特异性。结果构建了高效表达cd1-型亚硝酸盐还原酶的脱氮基因工程菌PNS。结论Cd1-型亚硝酸盐还原酶能够在脱氮基因工程菌PNS中得到正确和高效的表达。

Construction and expression of denitrification engineering bacteria for cd1 nitrite reductase

Objective To clone the nirS gene of Pseudomonas aeruginosa PAO1 into expression vector pQE-30 with the technology of gene engineering and express the nits gene effectively. Methods The primers were designed according to the nucleotide sequence of nitS gene published by GenBank and multiple cloning site （ MCS ） of expression vector pQE-30, then the nits fragment was amplified by polymerase chain reaction with the genomie DNA of Pseudomonas aerugi- nosa PAO1 as template. Following,the amplified product was digested by BamH I and HindⅢ ,forced cloning into pQE-30, transferred into DH5α and the transformant pQE30-nirS-DH5α was successfully constructed which included the recombination plasmid pQE3 transferred into the expression strain SG13009 to construct denitrification engineering bacteria pQE30 -nirS-SG13009（PNS）. Finally the molecular weight and specificity of cdl-nitrite reductase was identified by sds-PAGE（IPTG concentration ：0.05 mmol/L,induction temperature ：25℃,induction time ：2-3 h） and His-tag in-gel Stain. Result The denitrification engineering bacteria PNS which express cdl nitrite reductase effectively was constructed. Conclusion The cd1-nitrite reductase could express correctly and effectively in denitrification engineering bacteria PNS.