Description
Malaria is an infectious disease caused by the protozoan parasite, Plasmodium, transmitted by female Anopheles mosquito. According to WHO, in 2021, around 247 million cases of malaria were reported worldwide. When the parasite infects the host, there is a continuous reciprocation of metabolites between the two which may disturb the biochemical profiles of both. Hence, detection of the metabolites whose levels are altered in infected patients in comparison to healthy individuals may be a sign of parasite activity or the host’s response to the infection. For malaria detection, microscopy is the gold standard method and other available methods include polymerase chain reaction (PCR), rapid diagnostic tests (RDTs), and automated blood cell analyzers. So, several diagnostic methods are available for malaria but most of them are based on invasive methods and have limitations such as the pain associated, risk of contamination, and poor compliance in case of repeated sampling. Therefore, there is a need for an effective non-invasive method for malaria diagnosis. The methodology of this study includes sample collection, proteomics analysis by LC-MS/MS, cloning of selected proteins by PCR, purification and characterization of proteins, and ELISA based quantitative detection of proteins. The samples from P. vivax infected individuals and healthy individuals were collected and prepared (one method with SDS gel+in-gel digestion, second method with in-solution digestion and third method with short-SDS gel+in-gel digestion) for LC-MS/MS. In the mass spectrometry analysis, some Plasmodium proteins such as rifin, DBLMSP-2, merozoite surface protein, and GAPDH have been found in the urine samples of infected individuals. The proteins were screened based on half-life, dissimilarity with the human proteome, and abundance. The cloning of selected proteins is being done. For DBLMSP-2, PCR was performed but no gel bands were observed and then, primers were designed according to the domains. For one of the selected proteins, which has been reported in literature to be present in the urine of Plasmodium infected patients, the gene has been synthesised. The gene in pET-28a vector was transformed into E. coli BL-21 cells. The expression of the protein was checked by IPTG induction at 250 µM. The protein was expressed at this concentration and further processed for protein purification. SDS-PAGE was run to check whether the protein is present in soluble or insoluble fraction. The protein is in the soluble fraction and the protein purification will soon be carried out. The purified proteins will be checked as an antigen and as an antibody against the samples of Plasmodium infected patients using enzyme-linked immunosorbent assay (ELISA) to determine the diagnostic potential of these selected proteins for non-invasive diagnosis of malaria. The outcome of this project will be the identification of diagnostic biomarker proteins in the urine of patients infected with P. falciparum and/or P. vivax, which will help in developing better non-invasive diagnostic techniques for malaria.
Acknowledgements:
• Department of Biotechnology, Central University of Rajasthan
• School of Life Sciences, Central University of Rajasthan
• DST-INSPIRE.
• JLN Hospital, Ajmer
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