反义寡脱氧核苷酸对耻垢分枝杆菌抑制作用的研究

The Inhibition Efficacy Evaluation of Antisense Oligodeoxynucleotide Phoshorothioates Targeting Mycobacterium smegmatis

目的研究反义寡脱氧核苷酸对耻垢分枝杆菌生长的抑制作用，探讨其潜在的抗结核作用。方法在7H9培养基中接种5×10^5耻垢分枝杆菌MC^2 155，同时加入20μmol／L针对结核分枝杆菌中必须基因RV3806c在耻垢分枝杆菌中的同源基因MSMEG_6401的反义寡脱氧核苷酸。以耻垢分枝杆菌MC^2 155单独培养作为对照。观察和绘制细菌生长曲线；测定细胞膜MSMEG_6401编码的磷酸核糖转移酶的生物活性；应用高压气相色谱测定耻垢分枝杆菌细胞壁中糖的含量。结果与对照组相比，试验组耻垢分枝杆菌增殖速度平均降低0.45 OD（P〈0.01）；CFU计数平均延缓0．67Log；在应用等量的膜蛋白的情况下，实验组磷酸核糖转移酶的生物活性降低约40％，试验组和对照组细菌反应细胞壁的糖含量的指标甘露糖-半乳糖，阿拉伯糖比率没有明显差异（2组实验组分别为63％和69．4％；2组对照组分别为66．6％和69．1％）。结论针对MSMEG_6401的反义寡脱氧核苷酸对耻垢分枝杆菌的增殖有一定程度的抑制作用，推测反义寡脱氧核苷酸技术有一定抗结核价值；结核分枝杆菌的RV3806c基因或者是其编码产物可能是一个好的抗结核药物作用靶位。

Objective： To analyze the inhibition efficacy of antisense oligodeoxynucleotides targeting Mycobacterium smegmatis, and speculate on the potential anti-Tb effect of antisense oligodeoxynucleotides. Methods Designed and synthesized anti-ODNs targeting the MSMEG_6401 in M. Smegmatis which is the homologue gene of the essential gene Rv3806c in tuberculosis .Inoculated 20 μmol/L anti-ODNs and 5 × 10^5 cfu M. smegmatis MC^2 155 into 7H9 broth simultaneously, using the wild type smegmatis MC^2 155 as control. Monitored the growing curves of the test and control groups; after harvested the cells, analyzed the transferase activity of the membrane protein; analyzed the sugar components of the cell wall by Gas Chomatograhpy. Results Compared with the control group, the test group cell density was 0.45 OD less（P〈0.01）,and CFU counting lagged 0.67Log;When come to the mannose-glyctose/arabinose ratio, which indicated the sugar component quantity of the cell wall, there was no difference between test and control group（（The results for 2 test groups were 63% and 69.4% respectively, for 2 control groups were 66.6% and 69.1% respectively）. Conclusion The anti-ODNs targeting the MSMEG 6401 gene could inhibit the grow ofM. Smegmatis to some extent, makes it easy to predict that the anti-ONDs might inhibit M. tuberculosis too;Rv3806c gene itself and its encoding protein might be a good target for new TB drug development.