Slauch J M, Camilli A
Department of Microbiology, University of Illinois, Urbana 61801, USA.
Methods Enzymol. 2000;326:73-96. doi: 10.1016/s0076-6879(00)26047-3.
IVET was designed to identify those bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes encode virulence factors, products specifically required for the infection process. The paradigm IVET system is based on complementation of an attenuating auxotrophic mutation by gene fusion and is designed to be of use in a wide variety of pathogenic organisms. In S. typhimurium, we have used this system successfully to identify a number of genes that are induced in a BALB/c mouse and that, when mutated, confer a virulence defect. The RIVET system is based on recombinase gene fusions, which, on induction during infection, mediate a site-specific recombination, the product of which can be screened for after recovery of bacteria from host tissues. In V. cholerae, we have used this system successfully to identify genes that are induced transcriptionally during infection of the gastrointestinal tract of infant mice. RIVET is also uniquely designed for postidentification analysis of in vivo-induced genes: (1) it has been used to analyze the temporal and spatial patterns of virulence gene induction during infection and (2) it has been used to dissect the regulatory requirements of in vivo induction with respect to both bacterial regulatory factors and host-inducing environments. The IVET system has several applications in the area of vaccine and antimicrobial drug development. This technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live-attenuated vaccines. In addition, the identification of promoters that are expressed optimally in animal tissues provides a means of establishing in vivo-regulated expression of heterologous antigens in live vaccines, an area that has been problematic previously. Finally, we expect that our methodology will uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.
体内表达技术（IVET）旨在鉴定病原体感染宿主时被诱导表达的那些细菌基因。这些诱导基因的一个子集编码毒力因子，即感染过程中特异性需要的产物。典型的IVET系统基于通过基因融合对衰减性营养缺陷型突变进行互补，并且设计用于多种致病生物体。在鼠伤寒沙门氏菌中，我们已成功使用该系统鉴定了一些在BALB/c小鼠中被诱导且突变后会导致毒力缺陷的基因。重组体内表达技术（RIVET）系统基于重组酶基因融合，在感染期间诱导时，介导位点特异性重组，其产物可在从宿主组织中回收细菌后进行筛选。在霍乱弧菌中，我们已成功使用该系统鉴定了在幼鼠胃肠道感染期间转录诱导的基因。RIVET还专为体内诱导基因的鉴定后分析而独特设计：（1）它已被用于分析感染期间毒力基因诱导的时间和空间模式，（2）它已被用于剖析体内诱导相对于细菌调节因子和宿主诱导环境的调节要求。IVET系统在疫苗和抗菌药物开发领域有多种应用。该技术旨在鉴定毒力因子，因此可能会发现用作疫苗成分的新抗原。IVET系统有助于分离涉及毒力的基因突变，因此应有助于构建减毒活疫苗。此外，鉴定在动物组织中最佳表达的启动子提供了一种在活疫苗中建立体内调节的异源抗原表达的方法，这是一个以前有问题的领域。最后，我们预计我们的方法将揭示许多微生物在动物组织中生长所需的生物合成、分解代谢和调节基因。对这些基因产物的阐明应为抗菌药物开发提供新的靶点。
