Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, Listeria ivanovii (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing Mycobacterium tuberculosis antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the M. tuberculosis antigen gene Rv0129c into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the LIorfXYZ gene in the Listeria pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with LIorfXYZ at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish Listeria virulence without affecting its growth.

中文翻译：

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插入伊万氏李斯特菌基因组LIPI-1的异源基因的蛋白质表达水平取决于其插入方向。

由于它具有在吞噬细胞或非吞噬细胞中繁殖并随后引发强大的细胞免疫应答的能力，因此伊万氏李斯特菌（LI）被认为对于开发基于细菌的减毒活疫苗是可行的。我们以前生成了几种表达结核分枝杆菌抗原的重组LI菌株。由于有时异源蛋白的表达水平很低，我们试图阐明这种重组LI菌株中异源蛋白表达的原理。在这项研究中，我们将结核分枝杆菌抗原基因Rv0129c插入到LI菌株中，该菌株与基因组位于同一位置，但插入方向不同。RT-qPCR和Western blot结果表明，当异源基因的插入方向与细菌基因组中李斯特菌致病岛1中的LIorfXYZ基因相反时，异源基因可以被很好地转录，但蛋白表达水平受到限制。体外和体内。当以与LIorfXYZ一致的方向插入基因组中的同一位点时，在体外以及在小鼠模型中均观察到了预期的43 kD蛋白。重组后发现细菌毒力降低。这项工作证实了这种基于基因组重组的基于LI的疫苗中异源基因的蛋白表达水平与其在细菌基因组中的插入方向有关，并且在LIPI-1此位置插入的外源基因将消除李斯特菌毒力而不影响它的增长。