THE POTENTIAL OF CYSTEINE PROTEASE-ENCODING-GENE AS CANDIDATE FOR DNA VACCINE AGAINST PLASMODIUM BERGHEI INFECTION IN MICE
THE POTENTIAL OF CYSTEINE PROTEASE-ENCODING-GENE AS CANDIDATE FOR DNA VACCINE AGAINST PLASMODIUM BERGHEI INFECTION IN MICE
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Date
2013-01
Authors
BULUS, TIMOTHY
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Abstract
ABSTRACT
The potential of cysteine protease-DNA -vaccine- constructs to protect mice against Plasmodium
berghei infection was investigated. The full-length genes encoding the orthologs of falcipain-1
and falcipain-2 were identified in the database, isolated and amplified by a nested polymerase
chain reaction (PCR) using the genomic DNA of a rodent malaria parasite, Plasmodium berghei,
as template. The oligonucleotides used to prime the PCR were designed based on the identified
contigs at the Welcome Trust Sanger Institute containing the entire cysteine proteases reading
frames. The gel purified amplicons were cloned into pcDNA3AmpStrepTag vector and the
presence of the genes in positively transformed XL1-Blue E. coli strain were confirmed via
colony PCR, restriction digest and sequencing. The Berghepain-1 (BP1) and Berghepain-2
(BP2) genes amplified contain 1560 bp and 1407 bp open reading frames encoding 519 and 468
amino acids with molecular masses of 60.31 kDa and 54.46 kDa, and isoelectric points of 7.52
and 6.01, respectively. Sequence analyses and alignments showed that both deduced proteins
belong to peptidase_C1 superfamily, lacked signal peptides and possess high identity (up to
78%) with their corresponding cysteine protease orthologs from other malaria species and
conservation of the Cys, His and Asn residues that constitute the catalytic triad.
Immunoflouresence study with pcDNA3 plasmids harboring these genes and StrepTag
sequences, in frame, confirmed the transient expression of these proteins in COS-7-transfected
mammalian cells. The pcDNA3AmpBP1StrepTag and pcDNA3AmpBP2StrepTag midipreps
were cut with two sets of restriction endonuleases (BamHI/XhoI and HindIII/XhoI) for onward
ligation into pVax1 already cut with the same enzymes systems. The later strategies were
adopted to allow for the subcloning into pVax1 in order to produce
pVax1KanTransinBPStrepTag and pVax1KanBPStrepTag DNA vaccines for both enzymes
with and without transin signal peptide respectively. Mean total plasmid DNA produced with
transin was 2.73 mg while those without transin sequence was 3.02 mg. Seven groups of mice
(ten mice per group) were each immunized with the vaccine constructs and all the animals were
each sunsequently challenged with 1.43 x 107 P. berghei-iRBC. Mice in the positive and
negative control groups were given membrane-filtered PBS as placebo in place of DNA vaccine
and the groups were either challenged with the parasite or left unchallenged respectively.
Parasitaemia, animal weights and red cell counts were determined during the experiment. Deaths
were also closely monitored and survival rate of the experimental groups were noted. All values
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observed were statistically compared with values of the three control groups (Negative, Positive
and vector controls). Immunization with the DNA vaccine constructs lowered the parasitaemia in
mice vaccinated with pVax1StreptagBP2, pVax1TransinStreptagBP1, and
pVax1TransinStreptagBP2. This pattern in parasite burden also affected, in identical manner, the
red cell counts, mean weight and ultimately the percentage population of healthy survivors seen.
Animals vaccinated with bergheipain2 gene insert were better protected against the lethal
infection than those vaccinated with Bergheipain1-encoding plasmids. Better protection were
seen in groups of mice immunized with DNA vaccine with transin signal peptide than those
without it. The possibility of plasmid DNA integration into the genomic DNA of experimental
animals was also checked using PCR technique and results obtained did not show any possible
integration. This study supports the immunogenicity of plasmodium cysteine protease (CP) and
demonstrates the potential of bergheipain-based DNA vaccine to protect mice against infection
due to P. berghei. Though the protection appeared to depend on the homologue employed, it was
enhanced by the incorporation of signal peptide sequence at the 5′-end of the CP gene
Description
A DISSERTATION SUBMITTED TO THE POSTGRADUATE SCHOOL, AHMADU
BELLO UNIVERSITY, ZARIA, NIGERIA IN PARTIAL FULFILMENT OF THE
REQUIREMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF
PHILOSOPHY IN BIOCHEMISTRY
Keywords
POTENTIAL, CYSTEINE, PROTEASE-ENCODING-GENE, CANDIDATE, VACCINE, PLASMODIUM, BERGHEI, INFECTION, MICE