Transfecting Plasmodium berghei with the Plasmodium falciparum thiazole kinase gene

Abstract

As current antimalarial drugs become progressively ineffective due to parasite resistance there is need to develop and pursue new therapeutic strategies. Parasite-specific metabolic pathways promise to make an ideal source of novel drug targets and provide unique opportunities for chemotherapy. Vitamin B1 biosynthesis pathway in Plasmodium falciparum is one such pathway. The overall aim of this study was to transfect Plasmodium berghei to express the Plasmodium falciparum thiazole kinase (Pfthk) gene. Following successful DNA isolation and peR, a 909 base pair gene fragment was isolated from Plasmodium falciparum genomic DNA. Upon expression of the gene in bacterial systems, the protein was found to be soluble with a molecular weight of 34.67 kDa. The thk was successfully cloned into a P. berghei expression vector, pExpress-l thus generating a stable transfection construct. Parasites (2.5xl09) were obtained from Balb/c mice (with a 5% parasitaemia of schizont stage) and upon overnight in vitro culture, the schizont stage grew to 5.0x I09 parasites. For transfection of P. berghei blood-stage parasites, electroporation settings of I Kv, 25~lF and 200 Ohms were used. Time constants of 0.7 rns and 0.8 rns were attained for two samples, showing a successful electroporation. Analysis of data showed that PFTHK protein is soluble and the amino acid signal peptide analysis revealed that the protein was non-secretory and without a cleavage site. These are characteristics of an attractive drug target. Further studies in the development of the protein as a drug target are recommended.