Expression And Enzyme Activity Study Of Polyphosphate Kinase In Recombinant Bacteria PHT01-PPK/WB800N

Obejective: To provide a new method for the treatment of hyperphosphatemia of CKD-MBD,The recombinant plasmid p HT01-PPK containing polyphosphate kinase?PPK?gene was electrotransformed into Bacillus Subtilis WB800 N,which was induced to express the protein.And the enzyme activity was determined.Methods: 1.According to the gene sequence?2085bp?of polyphosphate kinase?Escherichia coli K¹2?known in Gen Bank?NC₀00913.3?,biosynthesis was carried out.After the primers designed,PCR was performed.And then the gene was digested with plasmid p HT01 respectively.Digested products were purified and recycled by Glue recovery purification kit.Later,these two products were ligated with T4 ligase to be p HT01-PPK,and transformed into E.coli DH5? by chemical methods.Positive transformants would be filtered by bacterium PCR,the double enzyme digestion and sequencing.2.The recombinant plasmids p HT01-PPK were electrotransformed into Bacillus subtilis WB800 N,which were screened and identified.The engineering bacterium p HT01-PPK/ WB800 N were added IPTG?1mmol/l?to induce protein expressing.And then these were analyzed by SDS-PAGE and Western Blot.3.The p HT01-PPK / WB800 N gene engineering bacteria,which could correctly express the target protein,were inoculated into LB medium containing specific concentration of phosphate and IPTG for 24 hours.And the phosphate concentration was measured before and after the reaction.Then the enzyme activity was identificated.Results: 1.The polyphosphate kinase gene?PPK?was successfully constructed and amplified by PCR.It was about 2085 bp after electrophoresis,which was consistent with the theoretical value.2.The recombinant plasmids p HT01-PPK was successfully constructed and transformed into Escherichia coli DH5?.After bacteria PCR,double digestion,electrophoresis showed PPK was about 2085 bp,which was consistent with the theoretical value.DNA sequencing showed that the sequence was consistent with sequence on Genbank?NC₀00913.3?completely.3.The recombinant plasmids p HT01-PPK were electrotransformed into bacillus subtilis WB800 N competent cells and protein expression was induced by IPTG.SDS-PAGE analysis showed that molecular weight of about 81 KD was found in the 1 m M IPTG-induced group compared with the untreated group,which was consistent with the theoretical values.Western Blot analysis showed that a specific band of molecular weight about 81 KD was seen in induction group p HT01-PPK / WB800 N,consistent with the theoretical value of the target protein.4.Detection of enzyme activity of genetically engineered bacteria p HT01-PPK/WB800N: compared with the groups p HT01/WB800 N and WB800 N,the phosphate concentration in the solution of p HT01-PPK/ WB800 N group was decreased and the difference was significant?P<0.05?,indicating that the genetically engineered bacteria had polyphosphate kinase activity.Conclusion: PPK gene was constructed into bacillus subtilis WB800 N successfully,which could express polyphosphate kinase and has enzyme activity.