Cloning, overexpression, purification and characterization of Plasmodium knowlesi lactate dehydrogenase.
Plasmodial lactate dehydrogenase, key enzyme of anaerobic glycolysis, has been shown to be a potential immunodiagnostic marker as well as a novel target for chemotherapy. We have cloned, overexpressed and immunochemically characterized the recombinant lactate dehydrogenase of Plasmodium knowlesi, the fifth human malaria parasite. The P. knowlesi lactate dehydrogenase (PkLDH) gene was PCR amplified and 0.9 kb PCR product was cloned into pGEM-T Easy vector. Sequencing and BLAST analysis revealed open reading frame of 316 amino acids of PkLDH showing 96.8% homology with Plasmodium vivax LDH and around 90% with Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale LDHs. The PkLDH gene was subcloned into pGEX-6P1 expression vector and the SDS-PAGE analysis revealed that about 70% of fusion protein was present in the soluble fraction. The fusion protein was cleaved with PreScission protease and recombinant PkLDH (34 kDa) was affinity purified to homogeneity. The purified PkLDH exhibited high reactivity with polyclonal and monoclonal antibodies against plasmodial LDH. The polyclonal antibody produced against purified recombinant PkLDH in rabbits showed high ELISA reactivity with both native and recombinant PkLDH and could detect parasite LDH in malaria infected blood samples by sandwich ELISA. The purified recombinant PkLDH can be used to produce P. knowlesi specific monoclonal antibodies for specific diagnosis of P. knowlesi infection in humans.
Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226001, India.
Vandana
V
Singh
Deep C
DC
Kaushal
Sushma
S
Rathaur
Niraj
N
Kumar
Nuzhat A
NA
Kaushal
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology; D000918:Antibody Specificity; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000097:blood / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D008288:Malaria / Q000097:blood / Q000175:diagnosis*; D008969:Molecular Sequence Data; D016790:Plasmodium knowlesi / Q000201:enzymology* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D015854:Up-Regulation
D000913:Antibodies, Protozoan; D011993:Recombinant Fusion Proteins; D007770:L-Lactate Dehydrogenase