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Development of Lactobacillus reuteri as a Vaccine Carrier
本計畫使用Lactobacillus reuteri發展乳酸桿菌載體疫苗, 其主要之考量為: ？本菌屬於廣泛宿主性之胃腸道正常菌叢, 可吸附於人及動物腸道之黏膜, 進行繁殖, 表現異源性蛋白抗原, 引發humoral immunity; ？疫苗給予採口服方式, 極為簡便、安全性高且成本低廉; ？本菌可產生廣效性殺菌物質reuterin, 舉凡原蟲、黴菌、細菌皆有殺滅之能力.故以本菌作為載體疫苗之研發, 對群體經濟動物之防疫將具有很好的效果.在計畫施行第一年, 將分成四大部分來進行, 分別為: ( 1 )自L.reuteri選殖可供構築表現分泌載體( expression secretion vector; ESV )之重要調控子( 如promoter, terminator及signal sequence ).( 2 )自其他乳酸桿菌之已知序列的調控子, 經以PCR合成後, 測試其在L.reuteri的表現功能.( 3 )豬瘟病毒E2基因之deletion分析, 以找出抗原決定位並進行免疫原性之測試.( 4 )豬萎縮性鼻炎毒素PMT ( P.multocida toxin )基因之deletion分析, 以找出抗原決定位並進行免疫原性之測試.其目的為構築最佳的ESVs, 並轉載具有最佳免疫原性之抗原基因.第二年為: ( 1 )L.reuteri表現分泌載體之構築, 異源性分泌酵素基因( 如levanase gene )之轉載及其表現分泌效力之測試; ( 2 )因應公共衛生之安全要求, ESV上之selection marker ( erythromycin resistance gene )將以levanase gene取代, 以inulin分解之狀態作為監控之依據; ( 3 )以構築好之ESV轉載上述來自細菌和病毒之抗原基因及其表現分泌效力之測試, 以瞭解經L.reuteri表現之抗原epitope, 是否確實能引發有效的中和抗體.第三年將進行in vivo動物試驗, 其工作項目分別為: ( 1 )分析洛德疫苗乳酸桿菌腸道分佈狀態; ( 2 )洛德疫苗乳酸桿菌安全評估; ( 3 )免疫後定期測定血中IgG及糞便中sIgA之力價; ( 4 )動物保護試驗, 以作為未來動物呼吸道與消化道系統免疫之用.
This proposal is categorized in the fourth section ( i.e.4.2and 4.5of the animal vaccine item: vaccines development of Hog cholera ( HC ), and important bacteria diseases )of the seven-important-research sections.To date, development of microorganism as vector vaccine has been carried out in four different categories which include the reconstituted virus-based vector, the attenuated gene-deleted virus vector, the attenuated Salmonella typhimurium vector, and the commensal lactobacilli vector.Disadvantages concerning with the former three categories are detailed in section 19of this proposal.The latter category, in addition to be exempted from those disadvantages of the former three categories, was found to bear additional advantages including: ( 1 )lactobacilli, being the normal flora of gastrointestinal ( GI )tract of animals, can easily adhere, replicate, express heterologous protein gene, and induce the humoral immune response of the host, ( 2 )application of vaccine can be achieved by the easy, safe, and economic oral-route, ( 3 )reuterin produced by Lactobacillus reuteri can kill many kinds of microorganism including protozoa, fungi, and bacteria.Consequently, development of L.reuteri as a vaccine carrier, as proposed in this project, would be quiet suitable for the massive immunization of the economic animals which are often raised in a large quantity of population.In order to construct the expression-secretion vector ( ESV )with high efficiency for use in L.reuteri as well as to clone the most immunogenic determinants from pathogens, four major topics scheduled to be finished in the first year of this project include: ( 1 )cloning the important ESV-regulator-elements ( i.e.promoter, terminator, and signal sequence )from L.reuteri genome, ( 2 )evaluating the possible use of the PCR-synthesized ESV-regulator-elements originating from the known sequences of other lactobacilli, ( 3 )analyzing the E2gene of HC by the deletion approach as to localize the most immunogenic determinant, ( 4 )analyzing the Pasteurella multocida toxin gene ( PMT )by the deletion approach as to identify the sections of sequence with the most immunogenic property.Another three topics coming up in the second year of this project will include: ( 1 )construction of L.reuteri ESVs and evaluation of the expression of levanase gene in the constructed ESVs, ( 2 )replacement of the antibiotic selection marker in ESVs with the enzymatic gene ( e.g., levanase gene ), ( 3 )cloning of E2and PMT genes respectively into the constructed ESVs and evaluation of their efficiencies of expression.Eventually, the in vivo animal experiments will be carried out in the third year as to evaluate the proficiency of our products.Four major topics scheduled to be finished in this year are: ( 1 )distribution-analysis of the constructed L.reuteri vector vaccines in the GI tract of swine, ( 2 )safety-evaluation of the constructed L.reuteri vector vaccines, ( 3 )titer-analysis of the seral IgG and fecal sIgA induced by the constructed L.reuteri vector vaccines, and ( 4 )protection-analysis of the application of the constructed L.reuteri vector vaccines followed with the challenge of pathogenes.In conclusions, eighteen major topics of experiment are scheduled to be accomplished in this project within three years ( for details please refer to the section 14of this proposal ).The final goal of this study is to provide the swine-field with three new vaccines against Atrophic rhinitis and HC.Furthermore, vaccines against versatile diseases of animals may be developed by the extension of the strategy used in this study.
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