Expression, Purification Of Plasmodium Falciparum Glutamate Dehydrogenase (GDH) And Preparation Of Its Monoclonal Antibodies

Malaria remains one of parasitic diseases endangering human health in the developing countries in tropical and subtropical regions. Early diagnosis of parasitemia is vital for the control of malaria. One reason that the morbidity and mortality of malaria is difficult to control is short of rapid, accurate and affordable methods for the diagnosis and treatment of malaria in epedimic ereas. The classical detections of malaria parasite, for example microscopy, QBC,PCR,are complicated experiment, labor intensive, requiring expensive apparatus and demanding expertise. These limitations, therefore, have prevented the widespread applications in remote areas. Recently, the immune chromatography assay based monoclonal antibody has made great success in diagnosis of malaria with rapid, fast and accurate result. Currently, the kits including ParaSight-F (Becton Dickinson, Cockeysoville, Maryland, USA), ICT Malaria P.f and ICT Malaria Rf/P.v (ICT Diagnostics, Sydney, Australia) and OptiMAL (Flow Inc, Portland Oreg), are capable of detecting malaria parasite in the field. However, these kits are still too expensive and still have limitations for widespread applications in the developing countries. Hence, a new diagnostic target antigen is urgently required.Plasmodium glutamate dehydrogenase(GDH) is one of important functional molecular in matter and energy metabolism and has a key role in carbon and nitrogen metabolism. GDH is expressed in the whole erythrocyte, which catalyze the NADP+-linked oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia, at the same time, changing oxidative coenzyme I II (NAD+,NADP+)to reductive ones(NADH ,NADPH).Previous studies have shown that plasmodium GDH is significantly different from that of vertebrate tissue in physical, chemical, biological and enzymatic properties. For example, the former which use only NADPH as cofactor is not affected by GTP or ADP in accordance with other coenzyme-specific enzymes, the latter which use both NADH and NADPH as cofactor is strongly inhibited and activated by GTP and ADP depending on the substrate concentrations. Clinicaland experimental evidences suggested that malarial parasites are more sensitive to oxidative stress than their hosts are. Enzymes of the parasite redox metabolism, as those of thiol metabolism and the NADPH-regenerating dehydrogenases, therefore, are vital to the survival and growth of malarial parasites. The GDH is assumed to be the major source of NADPH. What is more, the NADP+-specific GDH is absent from the host red blood cell. The GDH of FCCI/HN isolate exhibited 99% homologies in amino acid with that of Thailand. In addition, it has been suggested that the GDH exhibited 50% homologies with eryth eukaryotes, epiphyte, bacterium and 23% with human. Although there is no report GDH of gene for P.vivax, it may have high homologies with P.falciparum with evolution view .It is clear that the GDH will be a new target antigen diagnosis ofP.f/P.v simultaneously.In this study, the ultimate goal is to establish immune chromatography assay based GDH as diagnostic antigen which is be able to diagnose jR//P.v.Therefore, the monoclonal antibody which is against P.fa/P.v simultaneously is the key for the technique and the integral and three-dimensional stucture epitope with high biological activiey of GDH is the base. Based recombinant fusion expression plasmids, we cloned non-fusion expression plasmids. These plasmids were expressed in E.coli respectively. By renaturation and chromatography, the fusion and non-fusion protein were purified. Monoclonal antibodies were prepared against two proteins by hybridomas technology and identified. With colloid gold label and immune chromatography technology, the colloid gold immune chromatography was esbalished to estimate sensitivity and specificity compared to microscopic examination.The fusion express plasmid pGEX-4T-l/GDH expressed a 66KDa fusion protein in the form of inclusion bodies in E. coli, accounting up approximately 25% of total bacterial protein. The inclusion bodies were refold...