Johnston Christopher D, Bannantine John P, Govender Rodney, Endersen Lorraine, Pletzer Daniel, Weingart Helge, Coffey Aidan, O'Mahony Jim, Sleator Roy D
Biological Sciences Department, Cork Institute of Technology Cork, Ireland.
United States Department of Agriculture - Agricultural Research Service, National Animal Disease Center Ames, IA, USA.
Front Cell Infect Microbiol. 2014 Sep 4;4:120. doi: 10.3389/fcimb.2014.00120. eCollection 2014.
It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.
有充分文献记载，含有高GC含量的开放阅读框在富含A+T的宿主中表达不佳。具体而言，富含G+C的密码子使用是副结核分枝杆菌（MAP）蛋白在唾液乳杆菌中进行异源表达的限制因素。然而，通过同义替换对开放阅读框进行重新设计可以抵消密码子偏好，并大大提高该宿主中MAP蛋白的产量。在本报告中，我们证明对MAP2121c进行密码子使用操纵可以增强主要膜蛋白（MMP）的异源表达，类似于MAP杆菌天然产生的形式。当异源过表达时，单克隆抗体介导的ELISA表明合成的MMP蛋白中保留了抗原决定簇。此外，MMP是MAP中的一种膜蛋白，其在重组唾液乳杆菌的细胞表面定位水平与MAP相当。此外，我们之前对MAP3733c（编码MptD）进行了工程改造，在此表明MptD在共聚焦显微镜下显示出与细胞质膜边界相关的趋势，并且细胞内积累的蛋白选择性地粘附于MptD特异性噬菌体fMptD。这项工作表明唾液乳杆菌作为MAP的一种可行的抗原递送载体具有潜力，这可能提供一种有效的抗副结核的粘膜疫苗。
