Prokaryotic Expression And Purification Of GDH Protein And CDF Protein Of Clostridium Difficile

Objective: Clostridium difficile is one of the major pathogenic bacteriafor infectious diarrhea in hospitals. The Society for HealthcareEpidemiology of America（SHEA）and the Infectious Diseases Society ofAmerica (IDSA) released the Guidance for clinical diagnosis and treatmentspecifically targeting at CDI in2010. England has listed CDI as one of themandatorily reported and managed diseases. However, there is few reportson CDI in China, the reasons might be there are no fast and reliable detectionmethods and the available exported assay kits are expensive and withoutgovernment-approved documents. In foreign country, EIA is the mostcommonly detection used for CDI, detect GDH as screening, combineddetection of toxin A/B. GDH is the common antigen of CD and with its highoutput, GDH can be deteced sensitivity in short time so it’s often as aefficient screening method. New separation of high strain subtype ofBI/NAP1/027which produce the binary toxin (CDT) is considered to be themain cause of high morbidity and high mortality. The CDT includes twoseparate protein subunits: CDTa has the enzyme activity and CDTb has the combining ability. According to a new study the mortality rates aresignificantly higher in CDT merger TcdA and TcdB strains infection thanthat of TcdA and/or TcdB strains. It’s confirmed that the CDT test plays animportant role in CD clinical case management and in timely and effectivetreatment and in cure and disease outcome.Now there are not kits able todetect GDH and ToxinA/B in the same time. What’s more, there is no rapiddetection method of detecting the CDT. This study is designed to anaerobicculture the standard strains of CD, to clone the recombinant expressionplasmid of Clostridium difficile of GDH and CDTa/b by molecular clonetechnology, to prokaryotic express and purify the purpose protein, andto obtaine the monoclonal antibody of GDH and CDTa/b by hybridomatechnology, which can lay the foundation for rapid detection method of CDI.Methods: Anaerobic culture CD standard strain ATCC BAA-1805,extract genomic DNA of CD as PCR template.By PCR method, GDH andCDTa/b full length gene was amplified from template DNA and cloned intovector pET-28a(+) and pET-32a(+). The constructed recombinant plasmidswere transformed to E.coli Rosetta （DE3） for expression under inductionof IPTG. The expressed product was analyzed by SDS-PAGE and Westernblot and was isolated and purified by Ni-NAT. The concentration of enzymeactivity of GDH was determined by continuously monitoring method.Immune mice with purificated GDH, CDTa and CDTb, detect serumantibody titer in mice, and choose the mice with high titer to operate cell fusion to obtain monoclonal antibodies.Results: Successfully cultivate Clostridium difficile. Successfullyconstructed the recombinant plasmids pET-28-GDH,pET-32-CDTa andpET-32-CDTb. Purpose proteins are obtained by prokaryotic expressiontechnology. The products by SDS-PAGE analysis are visible that GDH inrelative molecular weight of about46kDa, CDTa in70kDa, CDTb in97kDa.Western blot analysis show that expressed products with a His tag. Thepurity of the protein GDH purified by Ni-NAT was more than90%andreached a concentration of1.7mg/ml. NADPH and NADP are coenzymes ofGDH and the concentration of enzyme activity of GDH were42.6U/L and63.3U/L. Proteins CDTa and CDTb accompanied by a small amount ofmiscellaneous protein after purification, the purity of the proteins CDTaand CDTb purified by Ni-NAT were70%and60%and concentrations were1.3mg/ml and1.0mg/ml. All mice produced antiserum after immunizationand the antiserm drop degree is1:105.Choose the mice immune with GDHand undertake the cell fusion but failed to sort out the successful integrationof hybridoma.Conclusion: The recombinant plasmid pET-28-GDH could expressstable and efficient in soluble foem and the purified protein with high purityand activity, which laid a foundation of developing monoclonal antibodies ofGDH. The recombinant plasmids pET-32-CDTa and pET-32-CDTbexpressed products are relatively small, and contained miscellaneous proteins. Proteins of CDTa/b can be further optimized in expression bychanging the temperature and concentration of IPTG and in purification byion exchange chromatography or by Superdex200molecular sieve affinitychromatography.