乳酸球菌Lactococcus lactis與枯草桿菌Bacillus subtilis一般皆被視為GRAS級安全的菌株，而利用安全的菌株所進行生產的醫藥產品是一般大眾所能接受的。所以本論文主要是研究利用被視為安全菌株的格蘭氏陽性菌-乳酸球菌與枯草桿菌做為生產醫藥品之宿主細胞，建立起其相對應之蛋白質表現系統，並利用此表現系統進行表現玻尿酸 (HA)相關基因產生玻尿酸，此外也利用此系統表現B型肝炎表面抗原(HBsAg)，並進一步研究在乳酸球菌表面表現此抗原。
在所建構能表現玻尿酸相關基因之乳酸球菌以乳酸鏈球菌素(Nisin)進行誘導可表現出HA，在利用葡萄糖與乳糖之培養基培養下，可生產0.68克/公升的玻尿酸。此外，將透明質酸合成之相關基因(HA synthase, hasA 與UDP-glucose dehydrogenase, hasB)與透明顫菌血紅蛋白(Vitreoscilla hemoglobin)以同源交換方式插入至枯草桿菌之染色體中，建構出可合成HA之Bacillus subtilis共同表現血紅蛋白，可大幅提升玻尿酸的生產量達2.01克/公升，此濃度與傳統所操作之病原菌Streptococcus equi subsp. Zooepidemicus所產之HA濃度相當。
此外，乳酸球菌也被用以進行疫苗蛋白表現之可行性的探討，B型肝炎表面抗原被用做目標蛋白來進行初步的研究，經由基因重組技術改質，所建構之乳酸球菌可於胞內表現出B型肝炎表面抗原；若再利用納豆菌Bacillus subtilis的表面蛋白pgsA與HBsAg融合，可利用此pgsA表面蛋白將HBsAg表現於乳酸球菌表面，如此將可大幅提升所表現之抗原與人體腸道細胞接觸的機會，增加其誘導出抗體之可行性。
由於最終使用於人體的基因改質菌體細胞，必須不含有任何抗生素與有害人體的基因，所以我們也成功地發展出利用乳酸鏈球菌素之免疫蛋白(nisin immunity protein, nisI)與threonine營養需求缺陷之質體修補系統，透過此兩質體系統的發展將可大幅提升利用乳酸球菌於表現醫藥用蛋白質及其代謝產物的安全性。

Generally recognized as safe (GRAS) bacteria Lactococcus lactis and Bacillus subtilis were genetically modified to make them have the capability to produce biopharmaceuticals such as hyaluroic acid and HBsAg. In addition to construct recombinant GRAS strains for biopharmaceutics production, food-grade vectors were also developed for being used in Lactococcus lactis to express the desired proteins.
Microbial hyaluronic acid (HA), commonly produced by pathogenic Streptococcus was produced by Lactococcus lactis by co-expressing HA synthase and UDP-glucose dehydrogenase (UDP-GlcDH) of Streptococcus equi subsp. zooepidemicus in a nisin controlled expression (NICE) system. With nisin induction, the recombinant L. lactis cultured in glucose (1%) supplemented M17 medium can produce HA to concentration of 0.65 g/L.
Hyaluronic acid (HA) producing Bacillus subtilis strains were also constructed by integration HA synthase gene (hasA) and UDP-glucose dehydrogenase gene (hasB or tauD) expression cassettes into the amyE locus of B. subtilis chromosome. All the inserted genes were under the control of a relatively strong constitutive vegII promoter of B. subtilis. HA production was enhanced at least 2 fold by co-expressing hasB or tauD with hasA. Vitreoscilla hemoglobin (VHb) was also co-expressed with the HA-expressing genes to increase HA production by B. subtilis. With the expression of VHb, not only the cell concentration was enhanced 25 % but also HA concentration was increased by 40 %. About 2.0 g/L of HA was obtained by the strain carrying VHb hasA, and tauD genes in the expression cassette after 32 h cultivation.
The food-grade Lactococcus lactis is a potential vector to be used as a live vehicle for the delivery of heterologous proteins for vaccine and pharmaceutical purposes. For safe and effective delivery of viral antigens to the mucosal immune system, a surface antigen display system, pRKNchbv, for lactic acid bacteria using the poly--glutamic acid synthetase A protein (PgsA) of Bacillus subtilis as an anchoring matrix have been developed and could have the target protein HBsAg displayed on the cell surface of L. lactis. the food-grade vector, pNisI-C that used nisin immunity protein, nisI as selection marker and nonantibiotic plasmid selection system pHT838C containing threonine biosynthetic enzymes, lactococcal theta-type replicon, and a P170 promoter-polylinker were also developed for L. .lactis to express biopharmaceutic protein and to produce metabolites that can be used in food.

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