Sequestrin of Plasmodium falciparum
AssigneeThe United States of America as represented by the Secretary of the Army
InventorsPatrick E. Duffy, Christian F. Ockenhouse
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Abstract
In this application is described the expression cloning and functional characterization of the CD36-binding domain of Sequestrin. Sequestrin is a surface protein of 215-250 kD size which does not vary in size between parasite strains. The Sequestrin gene appears to be single-copy-number within the genome; noncytoadherent laboratory parasite strains carry the sequestrin gene but fail to express the protein. Sequestrin binds with high affinity to CD36. Sequestrin protein and sequence can be used as a diagnostic, prognostic, and therapeutic tool.
Claims26
An isolated and purified DNA fragment which encodes a peptide of Sequestrin of Plasmodium falciparum, said DNA fragment comprising a DNA sequence encoding at least 5 consecutive amino acids of SEQ ID NO:2.
A DNA fragment which encodes a peptide of Sequestrin of Plasmodium falciparum, said DNA fragment comprising the sequence specified in SEQ ID NO:1, or a polynucleotide fragment of SEQ ID NO:1 comprising at least 15 nucleotides.
An isolated and purified DNA fragment comprising coding sequence for the peptide fragment of Sequestrin protein of Plasmodium falciparum specified in SEQ ID NO:2, or a polypeptide fragment of SEQ ID NO:2 comprising at least 5 amino acids.
An isolated and purified DNA fragment encoding a CD36-binding domain of Sequestrin of Plasmodium falciparum comprising the nucleotide sequence specified in SEQ ID NO:3.
A method for detecting the presence or absence of RNA of Sequestrin, a protein of Plasmodium falciparum, in a sample using the polymerase chain reaction, said method comprising:
- (i) extracting RNA from the sample;
- (ii) converting the RNA into complementary DNA (cDNA);
- (iii) contacting said cDNA with
- (a) at least four nucleotide triphosphates,
- (b) a first primer comprising at least 15 consecutive nucleotides of SEQ ID NO:1, and
- (c) an enzyme with polynucleotide synthetic activity,
- under conditions suitable for the hybridization and extension of said first primer by said enzyme, whereby a first DNA product is synthesized with said Sequestrin cDNA as a template therefor, such that a duplex molecule is formed;
- (iv) denaturing said duplex to release said first DNA product from said Sequestrin cDNA;
- (v) contacting said first DNA product with a reaction mixture comprising:
- (a) at least four nucleotide triphosphates,
- (b) a second primer comprising at least 15 consecutive nucleotides of the complement of the nucleotide sequence of SEQ ID NO:1, and
- (c) an enzyme with polynucleotide synthetic activity,
- under conditions suitable for the hybridization and extension of said second primer by said enzyme, whereby a second DNA product is synthesized with said first DNA product as a template therefor, such that a duplex molecule is formed;
- (vi) denaturing said second DNA product from said first DNA product;
- (vii) repeating steps iii-vi for a number of times sufficient to achieve linear production of said first and second DNA products;
- (viii) fractionating said first and second DNA products generated from said Sequestrin cDNA;
- and
- (ix) detecting the presence or absence of Sequestrin RNA in the sample by detecting the presence or absence of products generated from Sequestrin cDNA.
A malaria diagnostic kit comprising primers each primers comprising at least 15 consecutive nucleotides of SEQ ID NO:
- 1 specific for the amplification of DNA or RNA of Sequestrin, in a sample using the polymerase chain reaction and ancillary reagents suitable for detecting the presence or absence of Sequestrin DNA or RNA in a sample.
An isolated and purified polynucleotide encoding a CD36-binding domain of Sequestrin of Plasmodium falciparum comprising nucleotides encoding amino acids 42-180 of SEQ ID NO:2.
Dependencies are inferred from the claim text.