Prokaryotic Expression And Exploring The Regulatory Mechanism On The Acid Adaptation Of Regulator Protein Crdr From Helicobacter Pylori

Objective Prokaryotic expression regulator protein CrdR fromHelicobacter pylori26695standard strain to explore its role in acidadaptation regulation. From the physiological view of bacteria adaptation,it can supply the experimental materials and experimental basis to explorenew ways of anti-infective.Methods The whole genomic DNA of Helicobacter pylori strain26695was abstracted. The hp1365gene fragment was amplified with theabstracted genomic DNA as template by PCR. The amplified genefragments were cloned into the prokaryotic expression plasmid pGEX-6P-1.The recombinant plasmid was transformed to E.coli JM109and wasexpressed with inducing of IPTG. The expressed patterns of therecombinant protein were analyzed by SDS-PAGE and the expressionproducts were identified by Western blot and purified by affinitychromatography. The rabbit was immuned with the purified recombinantprotein CrdR to obtain the immune serum. The expression products of CrdR under different pH values were analyzed by Western blot to exploreits role in acid adaptation regulation.Results The PCR product is642bp consistent with the size of thehp1365gene; recombinant plasmid pGEX-hp1365was identified by doubledigestion and DNA sequencing, the inserted fragment and hp1365genesequences were identical. The relative molecular mass of recombinantprotein was approximately25KDa and its protein content was60%in theform of inclusion bodies via the SDS-PAGE analysis. Western blot canidentify the CrdR protein. The immune serum of the recombinant proteinCrdR was obtained by immunizing the rabbit. It can be showed fromWestern blot analysis that the highest expression of Helicobacter pyloriprotein CrdR in pH5medium condition and the expression was decreasedwith the increasing of pH.Conclusion The protein of CrdR was successfully expressed and itplays a role in Helicobacter pylori acid adaptation. It can supply theexperimental materials to further explore the mechanism of the CrdR andanti-Helicobacter pylori infection by blocking CrdR.