Cloning and recombinant expression of a 822 bp region of a Pf403 Plasmodium falciparum gene.
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Date
2003
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Abstract
Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium.
With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for
4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South
Africa has imposed severe costs on the economy and public health.
Immunity to malaria is a multi-component system involving both B and T celllymphocytes.
Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It
is known to be associated with the outer membrane of mouse erythrocytes infected with the
parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T
cell clone has been identified that adoptively provides protection to athymic mice infected with
P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the
proliferation of the protective T cell clones. At least four other antigens of different species of.
malaria share at least one cross-reactive epitope.
In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence
was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa
protein with a high degree of homology to Pc96. Sequence alignments indicated a region
spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa
protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope
prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in
Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and
is conserved in both sequences. PCR primers were designed to amplify regions of the
MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the
MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology
to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A
BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning
vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI
sites within the fragment.
The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning
kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV
complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion
analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI
site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of
the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose
binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa
protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by
anti-MBP antibodies, by western blotting. This recombinant protein has many applications for
further studies involving the characterisation of the Pf403 protein, and the determination of
possible roles that the protein may have in stimulating an immune response during human
malaria infections.
Description
Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.
Keywords
Plasmodium falciparum., Proteins., Malaria--Research., Malaria--Immunological aspects., Plasmodium yoelii yoelii., Cloning., Recombinant proteins., Malaria vaccine--Research., Theses--Biochemistry.