Vol 27 No 2 December - The Indian Society for Parasitology

JPD : Vol. 27 (2), 2003
AMA1 and DBP polymorphism in P.vivax
105
erythrocyte binding protein of P.falciparum shows sequence of R-II and R-VI) were amplified by PCR
homology. (Sim et al 1994). The sequence analysis of using specific primers. The sense and anti sense
duffy binding protein (DBP) of P. vivax allowed primers used for AMA-1 were: 5' AGA ATT CCA GCT
division of this gene into six domains named Region-I GGA AGA TGT -3' and antisense 5' TCC TAA ATT
to Region-VI based on amino acid sequences (Adams TTT ACG GGG GC-3. For DBP the sense and
et al 1992). Chitnis and Miller (1994) has identified the antisense primers were: 5' GAA GAT ATC AAT TAT
Region-II (N' terminal) as a critical ligand which binds GTA TG -3' and antisense 5' CTT CAA ATT CCT TTT
to host erythrocytes (Singh et al 2001). Thus, Duffy TCA TG - 3. For AMA-1, thirty five cycles of 95°C for
binding protein is essential for the survival of P.vivax. 1 min, 55°C for 50 sec and 72°C for 2 min, and for DBP,
30 cycles of 95° for 1 min, 48° C for 30 sec and 74° C
Due to the importance of AMA-1 and DBP in the life
for 3 min were performed. PCR products were
cycle of P. vivax both these antigens are strong vaccine
electrophoresed on 1.5% agarose gel and the fragment
candidates. As significant genetic diversity could
size was determined by comparison with molecular
compromise the efficacy of an otherwise effective
weight marker (100 bp ladder plus, MBI Fermentas).
vaccine (Crewther et al 1996), we investigated the
genetic polymorphism by PCR-RFLP techniques in The amplified products of AMA-1 and DBP gene were
AMA-1 and DBP in North Indian isolates of P.vivax. digested with Pvu-II and EcoR-I endonuclease
MATERIAL AND METHODS
respectively. Digestion was performed in a 20µl
reaction mixture utilizing 16 µl of PCR product,
The blood samples were collected from symptomatic 1.5 µl sterile ddH2O, 2µl (1 X) digestion buffer and
malaria patients found positive for Plasmodium vivax
0.5 to l µl (5 units) of restriction enzyme. The digestion
by microscopy. The patients included were those 0
was carried out at 37 C overnight. Finally, 10 µl of each
attending Nehru Hospital of Postgraduate Institute of
digested PCR product was electrophoresed on a 2%
Medical Education & Research, Chandigarh or agarose gel containing ethedium bromide and
malaria clinics and hospitals in various regions of
photographed in a UV transilluminator.
Northern India. The patients showing mixed infection
(with P. falciparum and P. vivax) were excluded. 2-3 All the 25 clinical isolates were amplified. No PCR
ml of blood was obtained from each patient by product was obtained in control samples which
venepuncture in a citrated anticoagulant (Laserson included the DNA extracted from P.falcipamm and
et al 1994).
blood samples from healthy individuals.
The DNA was prepared by a rapid method as describe RESULTS
by Foley et al (1994) with slightly modifications.
The PCR amplified products of AMA-1 and DBP gene
Briefly, 50µl of parasitized blood was washed thrice
produced a single band, the molecular weight of PCR
with 1 ml of ice-cold 5 mM sodium phosphate (pH 8.0)
products being approximately 500 bp and 2.3 kb
by mixing well by vortexing. Finally the suspension
respectively. No size polymorphism was observed on
was centrifuged at 10000 rpm for 10 minutes and 50 µl
electrophoresis in both the genes. (Figure-1&2)
sterile distilled water was added to the pallet and after
mixing was boiled for 10 minutes. The suspension was The RFLP patterns, (using Pvu-II and EcoR-l
centrifuged at 10000 rpm. Ten µl of supernatant was restriction enzymes for AMA-1 and DBP respectively)
used as DNA template in 50µl polymerase chain obtained did not show differences among these 25
reaction (PCR) mixture.
clinical isolates. The restriction digestion of PCR
products of AMA-1 gene from all the isolates resulted
All PCR reactions were carried out in a 50µl reaction in two fragments of 280 and 220 bp (Figure -1). The
volume in a thermocycler (Eppendorf master cycler restriction digestion of PCR product of DBP gene in
gradient). Amplification was performed in 50 mM all the 25 isolates resulted into two fragments of 1600
KC1, 10 mM Tris-Cl (pH 9.0), 1.5 mM MgCl 2 0.01% bp and 700 bp (Figure-2).
gelatin, 0.2 mM each of dNTPs, 1.25 unit AmpliTaq
Thus, the PCR-RFLP analysis of the HVR region of
DNA polymerase (GIBCO-BRL), 250 nM each of
AMA-1 gene and central polymorphic region of DBP
primers and 10 µl of parasite DNA as template. The
gene revealed that the P. vivax isolates did not show
Hypervariable region of AMA-1 and central
polymorphic region of DBP gene (Including partial
variation in the target genes and probably these were
conserved due to functional constraints.