PURA COMPLEMENTATION FOR PLASMID SELECTION & MAINTENANCE

活的减毒沙门氏菌菌株正在进行调查 携带外源抗原的载体刺激 针对各种细菌、病毒和 原生动物疾病。这些口服疫苗目前在 发育依赖于外源抗原的表达 也编码氨苄西林耐药性的载体。这不太可能 携带功能性抗生素基因的疫苗株将 为监管机构或广大公众所接受。 这项提议是为了研究替代方案的可行性。 一种选择系统，它应该选择性地将质粒保持在 没有任何外在施加的选择性压力。 细菌宿主染色体Pura缺陷的互补 活性腺基琥珀酸合成酶(Pura+)表达载体的构建 应该为保持质粒提供强大的选择 向量。这项研究将确定是否选择嘌呤 独立性可以取代标准的氨苯西林选择 小鼠伤寒沙门氏菌和都柏林沙门氏菌疫苗的小规模液体培养 菌株在实验室、10L规模发酵和体内 在小鼠模型系统中。选择性的成功示范 Pura质粒菌在培养和体内的维持 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.