Cloning, Expression Of The Fumarate Nitrate Reduction(FNR) Protein In Aeromonas Hydrophila XS91-4-1

Fumarate Nitrate Reduction protein is short for FNR. FNR, with another three global regulators-ArcA, NarL, and NarP, control the various pathways of respiration. As a key regulator in cell respiration, its role is to sense oxygen and determine whether the cell will grow aerobically or anaerobically, to allow the bacterium to adapt to changes in oxygen availability in its environment. A lot of research indicated that FNR is a protein containing Fe-S clusters. Under different oxygen conditions, Fe-S clusters play a central role in DNA-binding and transcriptional regulation.In invasive fish diseases, the bacterial disease caused by the pathogenic Aeromonas hydrophila has a substantial impact on aquaculture of our country. At present, the fishes are not only resistant to the drugs, but also caused serious pollution in the water body. It is important to find an efficient inhibitor to control the disease caused Aeromonas hydrophila. FNR, as a key target for regulating factor in Aeromonas hydrophila respiration regulation, is an ideal target that has specific effect on Aeromonas hydrophila. Studying the mechanism of FNR has an important significance for screening high-efficiency and safety inhibitor.In this study, gene engineering technology and prokaryotic expression system are used for the construction and expression of FNR, respectively. The DNA of FNR was amplified by polymerase chain reaction (PCR) with the genome of Aeromonas hydrophila XS91-4-1, which was used as a template. The full length of the FNR DNA was756bp, encoding252amino acids. The recombinant plasmid was constructed by subcloning the FNR gene into the prokaryotic expression vector (pET-28a) and then transformed into the host bacteria E. coli BL21(DE3) for expression. The SDS-PAGE analysis shows that: the recombinant plasmid was expressed at high level in E. coli BL21after0.25mmol/L IPTG induction and got the specific protein band which molecular weight was approximately32kDa. The effects of temperature and IPTG concentration on the FNR expression were also investigated and the results were listed below:30℃were chosen as the induced temperature; the protein expression was the best at0.25mmol/L IPTG. Target protein band could be observed when recombinant E.coli was induced beyond2hours and the amount of expressed protein reached its peak at4hours. After the ultrasonic breaking, SDS-PAGE analysis showed that the expressed protein existed mainly as soluble protein.In this paper, FNR gene was cloned and also expressed successfully, it could be used for the large scale preparation of high purity, strong activity of FNR, which can be used as a target enzyme for novel drug design in the future.