Center for Infectious Diseases and Vaccinology, The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.
Vaccine. 2010 Aug 23;28(37):6094-103. doi: 10.1016/j.vaccine.2010.06.074. Epub 2010 Jul 3.
The Salmonella rfc gene encodes the O-antigen polymerase. We constructed three strains in which we replaced the native rfc promoter with the arabinose-dependent araC P(BAD) promoter so that rfc expression was dependent on exogenously supplied arabinose provided during in vitro growth. The three mutant strains were designed to synthesize different amounts of Rfc by altering the ribosome-binding sequence and start codon. We examined these strains for a number of in vitro characteristics compared to an isogenic Deltarfc mutant and the wild-type parent strain. One promoter-replacement mutation, DeltaP(rfc174), yielded an optimal profile, exhibiting wild-type characteristics when grown with arabinose, and Deltarfc characteristics when grown without arabinose. In addition, when administered orally, the DeltaP(rfc174) strain was completely attenuated in for virulence in mice. The DeltaP(rfc174) mutation was introduced into attenuated Salmonella vaccine strain chi9241 (DeltapabA DeltapabB DeltaasdA) followed by introduction of an Asd(+) balanced-lethal plasmid to designed for expression of the pneumococcal surface protein PspA. Mice immunized with either chi9241 or its DeltaP(rfc174) derivative expressing pspA were protected against S. pneumoniae challenge.
沙门氏菌 rfc 基因编码 O-抗原聚合酶。我们构建了三个菌株，在这些菌株中，我们用阿拉伯糖依赖性 araC P（BAD）启动子取代了天然的 rfc 启动子，从而使 rfc 表达依赖于体外生长过程中外源提供的阿拉伯糖。这三个突变菌株通过改变核糖体结合序列和起始密码子来设计合成不同量的 Rfc。我们将这些菌株与一个同基因的 Deltarfc 突变体和野生型亲本菌株进行了多项体外特性比较。一个启动子替换突变，DeltaP(rfc174)，产生了最佳的表型，当用阿拉伯糖培养时表现出野生型特征，而当不用阿拉伯糖培养时表现出 Deltarfc 特征。此外，当口服给予时，DeltaP(rfc174)菌株在小鼠中的毒力完全减弱。将 DeltaP(rfc174)突变引入减毒沙门氏菌疫苗株 chi9241（DeltapabA DeltapabB DeltaasdA）中，然后引入一个 Asd(+)平衡致死质粒，以设计表达肺炎球菌表面蛋白 PspA。用 chi9241 或其表达 pspA 的 DeltaP(rfc174)衍生物免疫的小鼠能够抵抗 S. pneumoniae 的攻击。
