| Our lives are closely related with microorganisms. Traditional methods to detectmicroorganisms must be operated by professional people in special laboratory andneed48hours to get results. With the increase of people’s care for hygiene of food,pharmaceuticals and public areas, there is a need to develop fast and effectivedetection methods. Most research institutes and corporations apply North Americanluciferase to detect microorganisms. However, this luciferase has many shortcomingssuch as low stability, limited sensibility, short turnover and so forth. This study aimsto express Luciola italica luciferase in E. coli, which is more applicable than NorthAmerican luciferase.The gene of Luciola italica was synthesized firstly and then recombinantexpression plasmid of Luciola italica was constructed. Transformed the recombinantplasmid into E. coli BL21(DE3) to get engineered strain, E. coli BL21（DE3）/pET28a-Luc. After induction with IPTG, the soluble target protein was expressedsuccessfully. The luciferase was separated and purified through immobilized metalion affinity chromatography (IMAC). After optimizing expression conditions, therecovery rate is high to80%and recombinant luciferase has high specific activity,8×109RFU/mg. Then the factors determining stability, half life and intensity of lightwere measured. It turned out that the lowest amounts of ATP, luciferin (100mg/L),Mg2+(5mM) which can be measurable are10E-10mol/L,10μL,8μL respectively.The most appropriate reaction temperature is35℃and the PH of beast buffer is7.8.Following research above, this study investigated the effect of glycerol, PEG,trehalose, DTT, sucrose, BSA and betaine on stability of recombinant Luciola italicluciferase. The results indicated that the best amount of these reagents for stabilizingluciferase are glycerol20%, PEG5%, trehalose, DTT0.8mM, sucrose10%, BSAμg/mL and betaine10%respectively. Finally, this paper optimized the buffer toenlong the half life of luciferase. |
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