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