Dunstan S J, Simmons C P, Strugnell R A
Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3052, Australia.
Infect Immun. 1999 Oct;67(10):5133-41. doi: 10.1128/IAI.67.10.5133-5141.1999.
This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the DeltaaroAD mutant of Salmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding beta-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimurium in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more beta-galactosidase and luciferase in S. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the aroAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the pagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive trc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimurium as a vaccine vector.
本研究描述了三种体内可诱导启动子表达质粒的构建与分析，这些质粒包含pnirB、ppagC和pkatG，用于在肠炎沙门氏菌鼠伤寒变种（以下简称鼠伤寒沙门氏菌）的DeltaaroAD突变体中递送外源抗原。编码β-半乳糖苷酶和萤火虫荧光素酶的报告基因用于评估鼠伤寒沙门氏菌在体外对不同诱导刺激的反应以及在免疫小鼠体内的启动子活性比较水平。结果确定，在体外和体内，ppagC构建体在鼠伤寒沙门氏菌中指导表达的β-半乳糖苷酶和荧光素酶均多于pnirB和pkatG构建体。破伤风毒素C片段编码基因在鼠伤寒沙门氏菌（BRD509）的aroAD突变体中，在这三种启动子的控制下表达。用在pagC启动子控制下表达C片段的减毒鼠伤寒沙门氏菌[BRD509(pKK/ppagC/C frag)]口服免疫的小鼠产生了最高的破伤风类毒素特异性血清抗体反应。pagC启动子在体内的荧光素酶表达水平和体外的C片段表达水平似乎等同于甚至不低于组成型trc启动子检测到的表达水平。然而，用BRD509(pKK/ppagC/C frag)免疫的小鼠诱导产生的破伤风类毒素特异性抗体水平明显高于用BRD509(pKK/C frag)免疫的小鼠，这表明外源抗原表达的特定位置可能对免疫原性很重要。还对三种启动子/C片段表达质粒中的核糖体结合位点（RBS）进行了诱变。尽管对三种不同启动子元件中的RBS进行了优化，但通过减毒鼠伤寒沙门氏菌递送至小鼠时，C片段的体内表达水平和总体免疫原性并未受到影响。这些研究表明，体内可诱导启动子可能会增强免疫原性，并提高鼠伤寒沙门氏菌作为疫苗载体的效力。
