Expression And Analysis Of The Plasmodium Falciparum Merozoite Surface Protein MSPDBL1and MSPDBL2

Part1Cloning, expression of recombinant Plasmodiumfalciparum merozoite surface protein MSPDBL1and MSPDBL2by wheat germ cell-free protein synthesis systemObject: To express recombinant P. falciparum merozoite surface proteinMSPDBL1and MSPDBL2by wheat germ cell-free protein synthesis system.Methods: The target gene sequences encoding MSPDBL1and MSPDBL2arederived from PlasmoDB and the target fragments were amplified by PCR. Thefragments were cloned to pEU-His vector using the In-Fusion cloning method and therecombinant plasmids were transformed into E. coil DH5α cells. The recombinantplasmid was identified by DNA sequencing and the proteins were expressed in thewheat germ cell-free protein synthesis system. Finally, the recombinant protein wasanalyzed by western blotting and purified by affinity chromatography.Results: Six P. falciparum merozoite surface proteins rMSPDBL1(Mr74kDa),rMSPDBL2(Mr84kDa), rDBL1(Mr34kDa), rDBL2(Mr34kDa), rSPAM1(Mr17kDa),rSAPM2(Mr21kDa) were expressed successfully by wheat germ cell-free system,which were all soluble determined by western blotting.Conclusion: The wheat germ cell-free protein synthesis system can express P.falciparum merozoite surface proteins with high solubility. Part2Cloning, expression and immunogenic analysis ofMSPDBL2-DBL2domain from Plasmodium falciparumObjective: To clone and express the DBL domain of P. falciparum merozoitesurface protein MSPDBL2(DBL2), and investigate its antigenicity.Methods: The DBL2fragment was amplified by PCR and cloned into pET28avector. The recombinant pET28a-DBL2plasmid was transformed into E.coli BL21(DE3)cells and followed by expression of protein induced with IPTG. The expressed productwas purified by Ni-NTA affinity chromatography, and the expression was analyzed bySDS-PAGE and the antigenicity was investigated by western blotting.Results: The950bp of DBL2gene fragment was obtained by PCR. Therecombinant pET28a-DBL2plasmid was identified by PCR, double enzyme digestionand DNA sequencing. The recombinant DBL2protein was expressed in an inclusionbody form with Mr34kDa after inducing with IPTG. Moreover, the purified recombinantDBL2protein was recognized by the pooled sera from10P. falciparum exposedpatients by western blotting.Conclusion: The recombinant pET28a-DBL2plasmid has been constructed. Thepurified rDBL2exhibits good antigenicity and may be a potential candidate antigen P.falciparum. Part3The polymorphism analysis of Plasmodium falciparummerozoite surface protein MSPDBL2(Pfmspdbl2) inChina-Myanmar borderObjective: To analyze the allele polymorphism of the DBL domain of P.falciparum merozoite surface protein MSPDBL2(Pfmspdbl2) in China-Myanmarborder.Methods: PCR was used to amplify Pfmspdbl2, the PCR product of Pfmspdbl2was purified and sequenced, and the sequences were analyzed.Results: Totally16of Pfmspdbl2gene fragment was obtained by PCR among these samples revealed two genotypes, namely,3D7type and recombinant types, therate of3D7type was87.5%（14/16） and the rate of recombinant type was12.5%（2/16）.Conclusions: There exist polymorphisms for Pfmspdbl2in China-Myanmarborder.