Establishment Of Pichia Pastoris And Transgenic Medicago Sativa Expression System For Schistosoma Japonicum Antigen Sj23

Schistosomiasis japonica is a kind of parasite disease that seriously threats human's health, which also features the high rate of infection and recurrence. So far, it depends heavily on snail eradication and drug praziquantel for the prevention and treatment of this disease, but the effect is not very good. At present, vaccine has become a powerful weapon to control infectious diseases, so the development of cheap and effective vaccine for schistosomiasis japonicum has great economic value and social significance. Pichia pastoris and transgenic plants used as eukaryotic expression system have significant advantages of low price, safety and high efficiency when being used in the production of vaccine, which has become one of hotspots in the prsesnt acedemic study. In this study, Pichia pastoris and Medicago sativa expression systems for the antigen gene of Sj23 were established with the molecular biology methods to obtain protein vaccine and edible plant vaccine for Schistosoma japonicum. The achivements etails were shown as follows:(1) The Sj23 gene was amplified by PCR, and the recombinant yeast expression vector pPIC9K-Sj23 was constructed. After the recombinants expression vector was assayed by double digestion of restricted enzyme and sequenced, the result of sequences blasted with nucleotide sequence M63706 for the sequence of Schistosoma japonicum's antigen gene, the homology is to 99%. The results indicated recombinants expression vector pPIC9K-Sj23 had been constructed successfully.(2) The expression vector pPIC9K-Sj23 was transformed into Pichia pastoris GS115 by the LiCl method. The positive transformants were selected by PCR assay to obtain recombinant yeast strains. Then the recombinants yeast strains expressed the Sj23 gene in the culture medium was induced using methanol. When the culture supernatant was analyzed by SDS-PAGE, and a Sj23-expressed protein strip of 23KDa appeared, which indicated Sj23 gene is successfully transformed into Pichia pastoris and can be successfully expressed.(3) The fermentation conditions of recombinant strains were initially optimized in shake flasks. The results suggested that the optimum inducing time was 72 h, and the optimum pH was 5.0 when induced by 1.0% methanol at 30℃(4) LBA4404 (containing pCAMBIA1303-Sj23) was transformed into Medicago sativa through Agrobacterium–mediated method. Using the method of direct organogenesis, inducing the regeneration bud by the the culture medium of MS+2 mg/L 6-BA+0.5 mg/L NAA, and filtering the resistant regeneration bud through 50 mg/L Hyg,finally the regeneration plants were obtained in the rooted medium of 1/2 MS.(5) DNA were extracted from the regeneration plants and analyzed by PCR assay. The result suggested the gene Sj23 was integrated into the the transgenic plant genome.