PURA COMPLEMENTATION FOR PLASMID SELECTION & MAINTENANCE

减毒活沙门氏菌菌株正在研究中， 用于递送外源抗原以刺激免疫应答的载体 针对各种细菌、病毒和 原生动物疾病 这些口服疫苗目前 发展依赖于从质粒表达外源抗原 也编码氨苄青霉素抗性的载体。 可能性不大 携带有功能性抗生素基因的疫苗株 为监管机构或广大公众所接受。 这项建议是为了研究一种替代办法的可行性 选择系统，该选择系统应选择性地维持质粒， 不存在任何外部施加的选择性压力。 细菌宿主染色体中purA缺陷的互补 表达活性腺苷琥珀酸合成酶（PurA+）的质粒 应提供维持质粒的强有力的选择 vector. 这项研究将确定是否选择嘌呤 独立性可以替代标准氨苄青霉素选择， 小规模液体培养S.鼠伤寒沙门氏菌（S.都柏林菌苗 菌株在实验室中，在10 L规模发酵中，以及在体内 在小鼠模型系统中。 成功展示选择性 携带purA质粒的菌株在培养物和体内的维持 将导致purA质粒在活体口腔中的临床试验 细菌疫苗

Live attenuated Salmonella strains are being investigated as vectors for the delivery of foreign antigens to stimulate a protective immune response against various bacterial, viral, and protozoan diseases. These oral vaccines currently under development rely on expression of foreign antigens from plasmid vectors which also encode ampicillin resistance. It is unlikely that a vaccine strain carrying a functional antibiotic gene will be acceptable to the regulatory agencies or to the public at large. This proposal is to examine the feasibility of an alternative selection system which should selectively maintain the plasmid in the absence of any exogenously applied selective pressure. Complementation of purA defect in the bacterial host chromosome by a plasmid expressing active adenylsuccinate synthetase (PurA+) should provide a powerful selection for maintenance of the plasmid vector. This study will determine whether selection of purine independence could substitute for standard ampicillin selection in small scale liquid culture of S. typhimurium and S. dublin vaccine strains in the laboratory, in 10L scale fermentation, and in vivo in the mouse model system. Successful demonstration of selective maintenance of purA plasmid bearing strains in culture and in vivo will lead to clinical trials of purA plasmids in live oral bacterial vaccines.