Investigation Of Production And Immuno1-Protection Of Plasmodium Falciparum Major Merozoite Surface Protein 1

Malaria remains one of the most devastating infectious diseases in the world. The emergence of drug resistance of the parasite and development of resistance of Anopheles mosquito vectors demands a search for new tools to control the disease. Vaccination against the disease may be one of such tools which may control and even eradicate the disease from the world. Merozoite Surface Protein 1 of Plasmodium falciparum(PfMSP1) has been considered a leading candidate for malaria vaccine. The most convincing evidence of this view came from Aotus monkey trial in which immunization of the monkeys with native PtMSP 1 isolated from cultured falciparum malaria parasites provided partial to complete protection against the challenge of homologious strain. Moreover, rnonoclonal antibodies as well as polyclonal antibodies against the antigen inhibited the parasite growth in vitro. PfMSP1 is a glycoprotein of 190～200 kDa, synthesized during schnizogony. The molecule is proteolytically processed into four fragements, designated as MSP1-83, MSP1-31, MSP1-36 and MSP1-42. Synthesis of 5 kb redesigned PtMSPI gene of FCB strain and parts thereof resolved a major problem of the native gene with highly instability in heterogeneous system. It has been shown that the protection of PtMSPI required correct conformation of the protein. In this study, we carried out the production of the entire PfMSP 1 and parts thereof in Pichia pastoris as well as the study on their immunogenicity and protection against the parasite. 1. The 5 kb Pfmsp 1 synthetic gene was modified to eliminate the original signal peptide and to generate starting codon MG and Barn HI as well as Not Ⅰ restriction sites at 5'-and 3'-ends of the gene. The modified gene was inserted into pPIC3.5 expressing vector and the resulting plasmid was transformed into yeast P pastoris SMD1168 strain for cytosol expression. Our data showed that the entire Pfmsp 1 gene was successfully expressed in this system in cytosol form. The full-length of the protein was detected in the induced sample using monoclonalanibody mAb5.2, Whereas not deteCted in uninduced sample. The protein couldbe isolated by mAb5.2 affinity column, but it is difficuIt to prePare the protein atlarge scale for inununization.2. To identify any PouSP1 processed fragment which can induce proteCtiveimmunity, we attempted to produce all the processed fragments in Phoia ~Insertion of four PtheSPl processed fragments into pPICZa vector generatedrecombinant plasmid pPICZa/MSPl-83, pPICZa/MSPl-3l, pPICZa/MSPl-36 andpPICZa/MSPl-42. The plasmids were introduced into SMDll68 stfain fOrsecretory expression. Our results showed tha the MSPl-42 was exPressed insecreted form with doublet whereas the rest constructs gave no expression in thissystem. The MSPl-42 can be secreted into the protein-free medium. To measurethe confOrmational properties of MSPl-42 from the supematare, 15 monoclonalanibodies were utilized to interact with the PiChforderived MSPl-42. Of theseantibodies, ten are known to recognize conformational ePitope. The outcome ofthe interaction revealed that all antibodies specific for conserved and Kl protyPeinteracted with the protein whereas no ioteraction was observed betWeen theprotein and the monoclonal antibodies 9.7 and l2.l specific for MAD20.Interestingly, three monoclonal antibodies(e.g. 9.8, l3.l and 7.3) confirmedpreviously no interacion with CHO-derived MSPl can be interacted with thePiChforderived MSPl-42, implying the conformation of the prOtein derived fromN resembles more closely to the native protein.3. Rabbits were immunized with MSPl-42 recombinant protein fOrmulated withCFA aduvant with four times. The rabbits were bled on the dny 3 after lastimmuniza...