Outline. Background on Malaria and Plasmodium life cycle. Gateway cloning and using Expression vectors. Immune system, reverse vaccinology, and antibody study in mice. Future Studies. Malaria. Vector-borne infectious disease, caused by Plasmodium parasites P. falciparum – most lethal
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This uses “recombinational cloning” or the gateway method instead of traditional restriction digest and ligation into vectors.
Recombinational cloning is highly efficient and uses bacteriophage λ intergrase recombination proteins for directional recombination.
To isolate the Destination vector the mixture is introduced to E.coli by transformation and selected for.
Makes fusion proteins containing six histidines at the N terminus.
Upstream malEgene (maltose binding protein MBP). Results in a MBP fusion protein.
Protein of interest can be easily purified and isolated from MBP.
This may be due to toxicity of the expressed genes to E. coli or to the relative size of the expressed genes to those not expressed successfully.
Translates into reverse vaccinology
Chromosome 3 of
P. falciparumWhy Reverse Genetics?
ORF’s thought to code for membrane proteins