Key Laboratory of Animal Reproductive Physiology & Embryo Technology, Ministry of Agriculture, College of Veterinary Medicine, Northwest A & F University, Yangling, China.
Scand J Immunol. 2011 Nov;74(5):438-44. doi: 10.1111/j.1365-3083.2011.02596.x.
Tuberculosis caused by Mycobacterium bovis (M. bovis) seriously affects efficiency of animal production with impacts on public health as well. Effective programmes of prevention and eradication of M. bovis infection therefore are urgently needed. Intracellular pathogen resistance gene 1 (Ipr1) is well known to mediate innate immunity to Mycobacterium tuberculosis (MTB), but there are no reports as to whether Ipr1 can enhance the phagocytic ability of macrophage against M. bovis. In this investigation, RAW 264.7 macrophage was transduced with lentiviral vector carrying Ipr1 (named Lenti-Ipr1); transgenic cells were identified by RT-PCR and western blotting. Transgenic positive cells (R-Ipr1) were then infected with an M. bovis virulent strain, with non-transduced cells used as control. When cell proliferation, viability and apoptosis of the two groups were investigated, it was found that infected RAW 264.7 died by necrosis whereas R-Ipr1 underwent apoptosis. Furthermore, the numbers of intracellular bacteria in R-Ipr1 were lower than those in control cells (P < 0.05). To identify the role of Ipr1, we measured the genes of Casp3, Mcl-1 and NOS2A which associated with macrophage activation and apoptosis by real-time quantitative PCR. The results demonstrated that Ipr1 gene expression can enhance anti-M. bovis infection of macrophage. This establishes a basis for the future production of Ipr1-transgenic cattle to strengthen the tuberculosis resistance.
牛分枝杆菌（M. bovis）引起的结核病严重影响动物生产效率，同时也对公共卫生造成影响。因此，急需制定有效的牛分枝杆菌感染预防和根除计划。细胞内病原体抗性基因 1（Ipr1）已被证实可介导对结核分枝杆菌（MTB）的先天免疫，但目前尚无关于 Ipr1 是否能增强巨噬细胞吞噬牛分枝杆菌的能力的报道。在本研究中，用携带 Ipr1 的慢病毒载体转染 RAW 264.7 巨噬细胞（命名为 Lenti-Ipr1）；通过 RT-PCR 和 Western blot 鉴定转基因细胞。然后用牛分枝杆菌强毒株感染转基因阳性细胞（R-Ipr1），并以未转染的细胞作为对照。当研究两组细胞的增殖、活力和凋亡时，发现感染的 RAW 264.7 细胞坏死，而 R-Ipr1 细胞则发生凋亡。此外，R-Ipr1 细胞中的细菌数量低于对照组细胞（P < 0.05）。为了确定 Ipr1 的作用，我们通过实时定量 PCR 测量了与巨噬细胞活化和凋亡相关的 Casp3、Mcl-1 和 NOS2A 基因的表达。结果表明，Ipr1 基因的表达可以增强巨噬细胞抗牛分枝杆菌的感染能力。这为未来生产 Ipr1 转基因牛以增强结核病抗性奠定了基础。
