The Expression Plasmodium Vivax Malaria Transmission-blocking Vaccine Candidate Pvs25in The Silkworm Baculovirus System

Vivax malaria is an acute infectious disease caused by Plasmodium vivax, periodicallyattacks and is characterized with recurrent episodes at intervals of48hours. And now, due tothe emergence of the drug-resistant malaria parasite and the mosquito-borne resistant toinsecticide, the available antimalarial drugs meet new challenge. Preparation of a malariavaccine has become the primary way to effectively control spread of malaria.Transmission-blocking vaccine is a kind of mosquito stages of Plasmodium parasite surfaceprotein as an antigen-specific expression of the human body, to produce specific antibodyagainst mosquito stages of Plasmodium surface proteins. Vivax malaria transmission-blockingcandidate antigen Pvs25is the main target in the malaria research, which is highly conservedamong the strains of malaria in different areas.Pvs25is a kind of protein with molecular weight25kDa,which expresses on the surface ofzygote and ookinete, containing22cysteines that form11disulfide bonds, and its sequence hasfour epidermal growth factor-like domains (EGF-like Domain). In this study, we firstconstructed the prokaryotic expression plasmid of pET-32a-Pvs25, and then got the engineeringE.coli Rosetta bacteria. Induced by IPTG, the target recombinant protein was abundantlyexpressed in the bacteria. Moreover, after ultrasonication, the recombinant protein was in thecell supernatant. The target protein was purified by Ni affinity chromatography, the purity ofwhich is approximately95%.We utilized the purified recombinant protein to immunize Balb/c female mice to producepolyclonal antibody. The antibody titer measured by indirect ELISA was1:12800. Meanwhile,Utilizing the Bac-to-Bac baculovirus system of the silkworm bioreactor platform,wesuccessfully constructed the recombinant baculovirus vBm-Pvs25, which was used to transfectthe fifth instar larvae of Bombyx mori infected. According to Western Blotting analysis, therecombinant protein was expressed successfully. While constructing BmCMV-gp64-Pvs25, wecascaded CMV promoter in front of the polyhedrin promoter sequences in the vecter ofpFastBacDual, so that the expressed Pvs25could not noly be displayed on the surface ofBmNPV, but also be displayed in the mammals cells. We obtained the recombinant baculovirusvBmCMV-gp64-Pvs25, and transfected the BmN cells. Then, we identified the protein’s expression by SDS-PAGE, Western blotting, and Confocal scanning laser microscope. Theresults demonstrated that the target recombinant protein was successfully expressed in theplasma membrane surface. We used the aforementioned purified virus vBmCMV-gp64-Pvs25to immune Balb/c female mice to obtain antiserum, and the results show that the neutralizationtiter of the antiserum is1:25600, and the antigen can effectively stimulate the body to producethe protective immune response. This project establishes the foundation for further research ontransmission-blocking vaccine of vivax malaria.