Kanno Alex I, Goulart Cibelly, Rofatto Henrique K, Oliveira Sergio C, Leite Luciana C C, McFadden Johnjoe
Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, Brazil.
Programa de Pós Graduação Interunidades em Biotecnologia USP-IPT-IB, São Paulo, Brazil.
Appl Environ Microbiol. 2016 Apr 4;82(8):2240-2246. doi: 10.1128/AEM.03677-15. Print 2016 Apr.
The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
通过穿梭载体的开发，牛分枝杆菌卡介苗（Mycobacterium bovis BCG）或耻垢分枝杆菌（M. smegmatis）等分枝杆菌中许多抗原、刺激分子甚至代谢途径的表达成为可能，并且已经构建了几种重组疫苗。然而，这些系统中的任何一个中的基因表达大多依赖于通过反复试验选择预期能提供所需表达水平的天然启动子。为了系统地选择具有一系列强度的启动子，我们通过对最初来自分枝杆菌噬菌体L5的强PL5启动子进行易错PCR，生成了一个诱变启动子文库。这些启动子被克隆到增强型绿色荧光蛋白报告基因的上游，并鉴定出表现出广泛荧光水平的重组耻垢分枝杆菌。选择了一组启动子，并确定其在耻垢分枝杆菌和牛分枝杆菌卡介苗中具有高（pJK - F8）、中（pJK - B7、pJK - E6、pJK - D6）或低（pJK - C1）启动子强度。启动子区域的测序表明，在所有选定的质粒中它都被广泛修饰（6%至11%）。为了测试该系统的功能，证明了两种不同的表达载体能够在卡介苗中实现曼氏血吸虫抗原Sm29的相应表达水平。这里使用的方法可用于调整合成生物学和/或系统生物学研究的表达水平，或用于疫苗开发以最大化免疫反应。
