| Malaria remains one of the leading causes of morbidity and mortality in tropical and subtropical regions. It was estimated that 300~500 millon people were infected and nearly 3 millon were killed by this disease worldwide annually. Rapid, accurate and affordable methods are needed for the diagnosis and treatment of malaria. The microscopic detection of malarial parasite in the blood smear continues to be the most widely used technique for the diagnosis of malaria. However, the major limitation in its use is the time and expertise required for blood smear preparation and examination. Moreover, the primary health care institution are neither well equipped nor staffed with trained personnel. Although PCR-based tests are extremely sensitive, they are limited by the demands for considerable expertise and specialized equipment, which have prevented their widespread applications. Recently, the generation, antigen-capture tests are capable of detecting fewer parasites and of producing a result more rapidly and simply. The kits based on detecting histidine-rich protein- II (HRP- II ) and lactate dehydrogenase(LDH) of Plasmodium falciparum have applicated for malaria diagnosis in the field and made great success.Previous studies have shown that Plasmodium glutamate dehydrogenase(GDHp) is significantly different from that of vertebrate tissue in physical, chemical, biological and enzymatic properties. For example, the former which use only NADP(H) as cofactor is not affected by GTP or ADP hi accordance with other coenzyme-specific enzymes, the latter which useboth NAD(H) and NADP(H) as cofactor is strongly inhibited by GTP and activated or inhibited by ADP depending on the substrate concentrations. Epidemiological, clinical and 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 parasite GDH is assumed to be the major source of NADPH required for the reduction of glutathione disulfide(GSSG) by glutathione reductase. What is more, the NADP+-specific GDH is absent from the host red blood cell. In addition, it has been suggested that GDHp is likely to possess species- and genus-specific antigen, which is considered to be marker for the parasite compartment and a promising target molecule for the purpose of diagnosis and treatment. Since the enzyme is always present in current infection, studies are being carried out to use this enzyme activity to monitor the course of infections and the therapeutic response of patients with P. falciparum infection.In this study, we successfully amplified the gene encoding glutamate dehydrogenase from P. falciparum FCC1/HN strain by PCR. The PCR products were purified and cloned into the pGEX?T-l expression vector. Recombinant plasmids pGEX-GDH were screened and identified by PCR and restriction analysis. The cloned GDH gene was then sequenced by Sanger's method. DNA sequencing analysis showed that the size of gene was 1413bp, without any introns. By comparing with the GDH gene of Thailand isolate, it was found that there are only two point mutations occurred in the GDH gene of FCC1/HN isolate and these two deduced amino acid sequences are completely the same. Our results demonstrated that GDHp may be very conserved among different malarial species and strains. At the same time, GDH of FCC1/HN isolate exhibited 57.90%, 56.51%, 42.05% homology in amino acids with Giardia lamblia, Trypanosoma cruzi, Clostridium symbiosum, and 29.02%, 28.73%homology in amino acids with human GDH-1, GDH-2, respectively. It is very clear that Plasmodium GDH isobviously different from the GDH isozymes of other origins.The recombinant plasmid pGEX-GDH was transformed into E. coil BL21 and induced to express by IPTG. SDS-PAGE analysis suggested that the bacteria containing the recombinant plasmid produced a new fusion protein of 66KDa, consist...
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