Mycobacterium bovis BCG has been the only practical vaccine for tuberculosis. However, BCG cannot fully prevent adult pulmonary tuberculosis. Therefore, the improvement of BCG vaccine is necessary. We previously produced recombinant (r) BCG (BCG-PEST) for the better control of tuberculosis. BCG-PEST was developed by introducing PEST-Heat Shock Protein (HSP)70-Major Membrane Protein (MMP)–II–PEST fusion gene into urease-deficient rBCG using antibiotic-resistant gene for the selection of rBCG. HSP70-MMPII fusion protein is highly immunogenic and PEST sequence was added to enhance processing of the fusion protein. Although BCG-PEST effectively inhibited intrapulmonary growth of Mycobacterium tuberculosis (MTB), BCG with antibiotic-resistant gene is not appropriate for human use. Therefore, we produced antibiotic-resistant gene-free rBCG. We generated leucine-biosynthetic gene (leuD)-deficient BCG and introduced the fusion gene with leuD as the selection marker and named this rBCG as BCG-LeuPH. BCG-LeuPH activated human naïve T cells of both CD4 and CD8 subsets and efficiently inhibited aerosol-challenged MTB in mice. These results indicate that leuD can replace antibiotic-resistant gene for the selection of vaccine candidates of rBCG for human use.

牛分枝杆菌卡介苗是唯一实用的结核病疫苗。然而，卡介苗并不能完全预防成人肺结核。因此，卡介苗疫苗的改进是必要的。我们以前生产重组 (r) BCG (BCG-PEST) 以更好地控制结核病。BCG-PEST 是通过将 PEST-热休克蛋白 (HSP)70-主要膜蛋白 (MMP)–II–PEST 融合基因引入脲酶缺陷型 rBCG 中使用抗生素抗性基因选择 rBCG 而开发的。HSP70-MMPII 融合蛋白具有高度免疫原性，并添加了 PEST 序列以增强融合蛋白的加工。虽然 BCG-PEST 有效抑制结核分枝杆菌的肺内生长(MTB)，具有抗生素抗性基因的卡介苗不适合人类使用。因此，我们生产了无抗生素抗性基因的 rBCG。我们生成了亮氨酸生物合成基因（leuD）缺陷型BCG，并引入了以leuD为选择标记的融合基因，并将该rBCG命名为BCG-LeuPH。BCG-LeuPH 可激活 CD4 和 CD8 亚群的人类幼稚 T 细胞，并有效抑制小鼠中气溶胶攻击的 MTB。这些结果表明，leuD可以替代抗生素抗性基因，用于选择人用rBCG候选疫苗。