The Reaserch Of Recombinant Luciferase And Its Stability

Luciferase, a kind of high-efficiency biocatalysts is combosed by a polypeptide chain. Under condition of existing of ATP,Mg2+,O2 , the luciferase can catalysis luciferin to send out green light. With all the features above, the luciferase plays an important role in many fields.The target of this thesis is to realize domestic-scale production of the reconstituted luciferase and to research the stability of it. In my experiment, the gene of luciferase is amplified through technology of PCR and then the gene is linked with an expression vector, pET28a. The high-efficiency expression vector is achieved after the verification of cutting of restriction enzyme and sequencing. Luciferase is expressed successfully through the inducing of 1mmol IPTG during the fermentation and inducing conditions are optimized. The best inducing conditions are as follows: temperature 22℃, density of cell OD600 0.6, duration of inducing 18h, density of revulsive 1mmol. Based of results above, we attempt to expand the fermentation to pilot-scale. In the experiment, collected wet cell is broken through ultrasonic wave under the low temperature and the supernatant is recovered after centrifugal. The activity of luciferase in supernatant is detected by the rapid test device of ATP. Finally, 50ml pure luciferase is separated through Ion-exchange chromatography and affinity chromatography from 4g wet cell and its density is turn out to be 2.5mg/ml through the way of Braford. Our technologies introduced above are different from those who apply the way of renaturation of inclusion body, but recovery is improved in our experiment while cost is reduced. Then this thesis makes a deep discussion toward the conditions which influence the luminescence-producing reaction. Based on the results above, we research the sability of luciferase systematicly and create some protective solutes owing excellent performance price ration. We also attempt to apply the luciferase to rapid test of bacteria through desvising the test device and bacterial lysate, which can match with other reagents.