Construction And Expression Of The Recombinant Bacteria PAX01-PPK/WB800N Of Phosphorus Removal

Obejective:To provide a new idea for the treatment of chronic renal failure patients with hyperphosphatemia,We constructed therecombinant plasmid PAX01-PPK containing polyphosphate kinase(PPK)andtransformed into Bacillus Subtilis WB800 N,studyingits expression and the enzyme bioactivity.Methods:1.According to the Gen Bank data of Polyphosphate kinase(Escherichiacoli str.K-12)′s gene sequence,by biosynthesis,get the gene fragment of PPK.2.Design specific primer according to the genesequence,PCR amplification,recycling and depuration.The target fragment PPK and the plasmid PAX01 were double enzyme digested and connected,getting the recombinant plasmid PAX01-PPK.which were transformed into E.coli DH5 a,screening and sequencing identificating the transformant.3.Extract the recombination plasmid PAX01-PPK that has been cloned in E.coli DH5 a successfully,transforming into Bacillus Subtilis WB800 Nby electrotransformation,at the same time,screeningand identificating the recombinant bacteria.4.Addthe xylose to the recombination bacillus subtilis PAX01-PPKinduce the combinant protein PPK expression,meanwhile,compared with the control group,analyzing and identifing the expression products by SDS-PAGE and Western Blot.5.Preparing standard solution for phosphorus,determinate the optical densityand draw the standard curved line of phosphorus respectively.Engineering bacteria of the PAX01-PPK/WB800 N were cultured at a particular concentration of phosphate in the induced culture medium for 24 hours,set up the control group,and extract the culture medium to identify the enzyme activity by optical density.Results:1.The recombination E.coli DH5 acontaning of the PAX01-PPK were amplifide by bacterial PCR,double enzyme digested and sequencing identificated,the results were correspond with the target sequences.2.The recombination plasmid PAX01-PPKweretransformed into Bacillus Subtilis WB800 N by electrotransformation successsfully,the use of bacterial liquid PCR identification got a fragment about 2.085 Kb,the same with the target gene.3.The results of SDS-PAGE: the recombinant Bacillus Subtilis PAX01-PPK/WB800 Nexpressed protein that were induced,its molecular weight wasabout 81 KD,consistentwith the target fragment.Western Blot results displayed that a specific fragment appeared at 81 KD place,as the same as the size of expected protein.4.Results of enzyme activity detection: the standard curved line of phosphorus was drew.after 24 hours,constrast with the control group,the concentration of phosphate of theexperimental group was decreased,it reveled that the recombinant protain has polyphosphate kinase enzyme activity.Conclusion:Successfully construct recombinant Bacillus subtilis PAX01-PPK/WB800 N which can induce the expression of PPK,and the protein has the bioactivity.