Prokaryotic Expression And Identification The Role Of Regulation In Acid Adaptation Of Sensing Protein CrDS From Helicobacter Pylori

Objective: The sensing protein CrdS in the signal transduction s-ystem of Helicobacter pylori was prokaryotic expressed and analyzedwith bioinformatics to approach its regulatory function to the acidadaptation by the culture experiments of Helicobacter pylori in the ac-id experiment.Methods: The whole genomic DNA of H. pylori strain26695wasabstracted and set as the template.The hp1364genes coding CrdS wereamplified via the PCR technique.Then pUCm-T-hp1364of the clonal r-ecombinant plasmid and the prokaryotic expression plasmid pQE30-hp1364were built and identified by the ways of PCR, digesting with twoenzymes and sequencing.After that the expression plasmid pQE30-hp1364was transferred into the E.coli XL1-blue and induced with IPTG toexpress the protein CrdS.The molecular weight and the expressed formof the protein were analyzed by the method of SDS-PAGE.The express-ed protein was identified by Western blot and analyzed with the bioinf- ormatics.The rabbits were immuned with the purified recombinant prot-ein CrdS to obtain the immune serum.Through the experiments of H.p-ylori cultured in the vitro acid environment containing the immune se-rum of CrdS, the function of CrdS can be preliminarily identified.Results: The hp1364gene amplification product which templatewas the extracted genomic DNA was analyzed by agarose gel electr-ophoresis analysis.There was a visible clear strip in1200bp, and itsmolecular size was consistent with the hp1364gene. After the reco-mbinant cloning plasmid pUCm-T-hp1364and prokaryotic expressionplasmid pQE30-hp1364digested by BamHⅠ/HindIII, its products we-re analyzed by agarose gel electrophoresis analysis.There was a visib-le clear strip in1200bp.The secquence was measured by NCBI BLAST online and its result was exactly correct.The recombinant clonal p-lasmid pUCm-T-hp1364and the prokaryotic expression plasmid pQE30-hp1364was built correctly.It can be seen through SDS-PAGE analysisthat the expressed recombinant proteins induced with IPTG existed ma-inly in the form of the inclusion bodies and its relative molecular masswas about46kDa.The nucleic acid and amino acid sequences of CrdSwere exist steeply homology to other strains. Many hydrophilia district-s were presented on the surface of CrdS which contain many kinds ofeasily phosphorylated amino acids.The immune serum of the recombin-ant protein CrdS was obtained by immunizing the rabbits. Under diffe- rent pH conditions, the CrdS can affect the growth of H.pylori.Conclusion: The recombinant clonal plasmid pUCm-T-hp1364andthe prokaryotic expression plasmid pQE30-hp1364were built correctly andthe CrdS protein of H.pylori was expressed successfully. The protein takespart in the acid adaptation of H.pylori.It will provide the subject and basisof new way to resist the infection of H.pylori.