Studies And Applications Of DNA Vaccine Against Schistosome Japanicum

Schistosome Japanicum is a worldwide problem, which endangers the human health and even affects the development of civilization. Nucleic acid vaccine technology provides a new approach to prevent schistosomiasis and significant progress has been made towards the development of DNA vaccine against the infection. In this study, the antigen-coding genes of Schistosome Japanicum are cloned; gene-fusion of these antigen-coding genes has been achieved by recombinant PCR; DNA vaccines against Schistosome Japanicum are constructed; optimization of plasmid fermentation and processing skills of plasmid purification have been studied; the possibility of DNA vaccines'expression in both vivo and vitro is investigated. The main content was as following:1) Gene-clone of sj23, sjFABP, sj26, and sjGAPDH, which encode corresponding antigens respectively;2) Gene fusion of sj23 and sjGABP, as well as gene fusion of sj26 and sjGAPDH are accomplished by recombinant PCR. The successful fusion-genes are: sj23.FABP, sj26.GADPH;3) Sub-clone 4 types of antigen-coding genes of sj23, sjFABP, sj26, sjGAPDH and 2 types of fusion-genes of sj23.FABP, sj26.GADPH into expression vector pVIVO2 purposely; 14 kinds of DNA vaccine against Schistosome Japanicum are constructed;4) Fermentation conditions are optimized: glycerol is screened out from 5 kinds of carbon sources and soybean cake's extract is screened out from 6 kinds of nitrogen sources; the best proportion of K2HPO4 as additive is 20%; 3 important influencing factors of soybean cake's extract, beef broth and glycerol from 6 components of fermentation medium by"Plackeet-Burman"trials; the best composition of culture media is derived from"Box-Behnken"design (m/v) NaCl 1%,:Yeast Extract 1%, phosphate 0.3%( K2PHO4:KH2PO4=1:4), glycerol 1.68%, beef broth 2.28%, soybean cake's extract 4.21%, initial pH7.0.5) Preparation technology of DNA vaccine was set up: remove RNA with group gel filtration; remove open-coiled plasmid DNA with anion-exchange chromatography; remove endotoxin by affinity adsorption. The purified product was checked: no visible RNA, 0.11% protein within, 93.4% super-coiled plasmid DNA within, 0.85EU/(mg plasmid) endotoxin within. These norms successfully meet the requirement of DNA vaccine by SFDA.6) 14 kinds of DNA vaccines against Schistosome Japanicum are successfully expressed at the level of antigen in both muscle and cell line MCF-7 by IFA.