| Serratia marcescens nuclease(SM nuclease)is a kind of saccharide nonspecific endonuclease which can degrade all types of nucleic acid including single chain,double chain,linear and circular DNA or RNA,but without protease activity.According to the serial number M19495.1 of SM nuclease gene sequences posted on the NCBI website,removing its N-terminal signal peptide sequences,using E.coli's preference codons,designing and producing Ben-pGH with nuclease genes by synthesis.We used full length nuclease genes as the template and amplified them by PCR and then linked up with the pATa vector which was digested by NcoI and XhoI.The recombinant plasmid was transformed into E.coli Top10.We identified positive Ben-pATa clones by PCR and confirmed that prokaryotic expression vector Ben-pATa was successfully constructed through DNA sequencing.By blasting,it showed that the nuclease genes of Ben-pATa had 82%identities with the reported SM nuclease genes.Then we transformed Ben-pATa into E.coli BL21(DE3)and it could express recombinant nuclease in the form of inclusions under the action of IPTG.The paper optimized the best fermentation conditions in 250 mL shake flask,namely,choosing TB culture medium,5%inoculum density,100 m L loading volume,0.50mmol/L induced concentration of IPTG and 37?induced temperature.We tried it for large scale production.The paper also studied the different washing conditions of inclusions.The final washing condition was 100 mmol/L tris,2 mol/L urea,5 mmol/L EDTA and 1.5%Triton X-100 dissolved in the solution of pH8.0.After purifying and renaturing,the recombinant nuclease could effectively degrade the nucleic acids and it didn't have protease activity.The activity of the recombinant nuclease was 3.33×10~5U/mg,which was closed to other reported reports.Finally,the application of recombinant nuclease was also researched.It found that the bacteria liquid viscosity was reduced so that it could decrease the high pressure homogeneous resistance. |
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