The Expression And Preliminary Study Of Protein Pys25and Protein Pys48of Plasmodium Voelii In E.coli And BmN Cell

Yoelii malaria is caused by Plasmodium yoelii17XL, it occurs in Africa and its host ismouse. As different types of Plasmodium often have similar antigens, its primary structure andhigher formation have high homology, so it usually used as a model of human malaria in lab,particularly in immune response.The antigens Pys25and Pys48are produced in sexual reproduction period, Pys25is ansurface antigen exist in ookinete stage and Pys48is an surface antigen exist in gamete stage,there structures were conservative, and earlier studies shows that there antibody have obviouseffects in immune blocking test.We choose the two antigens for study, prokaryotic expression them in E.coli engineeringbacterias and eukaryotic expression them using surface display techniques in silkworm cells.Then we immuse Bacb/c mice, using the expressed proteins, The purpose of these experienceswere to identify whether the antibody gernerated by silkmorm cells were more effective inimmune blocking test, to testing whether the antibody generated by two antigens together weremore effective in transmission-blocking experiences, and to lay the foundation of later immuneblocking test and multivaccine, and to build a rodent model for othe rmalaria.First， we constructed the prokaryotic expression recombination engineering bacteriaBL21-pGEX-4T-1-Pys25and Rosseta-pET-32a-Pys48, then induce them by IPTG to generate thefusion proteins we want, then we collecting the bacterias, use high pressure to crush them,centrifuge them, collect the liquid supernatant and precipitate, we purified the protein Pys25byGST affinity chromatography; but the protein Pys48nearly insoluble, so we sissolved it ininclusion body solutions, We utilized these proteins to immunize Bacb/c mice to producepolyclonal antibody, measuring their titers are all up to1:12800by indirect ELISA. While, weuse the silkworm baculovirus surface displaying technology, insert mammal promoter CMVbefore Ph promoter in pFastBac Dual plasmid, construct the recombinant baculovirus vBmPys25and vBmPys48, and express the target proteins in silkmorm cells, immunofluorescence showsthat the target proteins were successfully displayed in BmN cells surface. Then we purified therecombinant baculovirions by ultracentrifugation(50000rpm40min), using them to immunizeBalb/c mice to get antibodies; these experiences lay the foundation of later immuse experiencesand provide some reference for other malaria.