反义寡脱氧核苷酸对耻垢分枝杆菌抑制作用的研究  

The inhibition effect of antisense phoshorothioate oligodeoxynucleotides on Mycobacterium smegmatis

作　　者：黄海荣[1] 赵雁林[1] 张宗德[1] 于霞[1] 刘冠[1] 

机构地区：[1]北京市结核病胸部肿瘤研究所,北京101149

出　　处：《中华检验医学杂志》2009年第12期1405-1408,共4页Chinese Journal of Laboratory Medicine

摘　　要：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 CFU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anti-ODNs作用6d后的耻垢分枝杆菌平均吸光度值（A值）为0.84±0.09；而野生型耻垢分枝杆菌平均A值为1.27±0.01，试验组耻垢分枝杆菌较对照组增殖速度平均降低0.45（P〈0.01）；anti-ODNs作用6d后耻垢分枝杆菌平均CFU计数的对数值为8.27±0.19；而野生型耻垢分枝杆菌平均CFU对数值为8.89±0.14，试验组耻垢分枝杆菌较对照组增殖速度平均降低0.62个log单位；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映细胞壁糖含量的指标甘露糖-半乳糖／阿拉伯糖比值没有明显差异（2组实验组分别为0.63和0.69；2组对照组分别为0.67和0.69）。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 effect of antisense oligodeoxynucleotides（anti-ODNs） on Mycobacterium smegmatis. Methods Synthesized anti-ODNs targeting the gene MSMEG 6401 in Mycobacterium smegmatis which is the homologne of the essential gene Rv3806c in Mycobacterium tuberculosis. 20μmol/L anti-ODNs were added into 5×10^5 CFU Mycobacterium srnegmatis MC^2 155 7H9 broth, with wild type of Mycobacterium smegmatis MC^2 155 as control. Growth curves of the test and the control groups were observed. After the cells were harvested, the ribose phosphate transferase activity of the membrane protein was analyzed. The sugar components of the cell wall were examined by gas chromatography. Results After 6 days＇ incubation, the average absorbanee values of cell density for the test groups and the control groups were 0. 84±0.09, and 1.27±0.01, respectively. The cell density of the test groups was 0.45 lower than that in the control groups （ P 〈 0.01）. The logarithm of the average CFU counting value for the test groups and the control group were 8.27±0.19 and 8.89±0.14 respectively. The cell density of the test groups was 0.62 log lower than that in the control groups. The bioactivity of phosphoribosyltransferase in the test groups was decreased by 40% with the existence of equivalent membrane protein. For the mannose-galaetose /arabinose ratio, which indicated the sugar component quantity of the cell wall, there was no significant difference between the test groups and the control groups （ The results for the 2 test groups were 0.63 and 0.69, respectively, and the results for the 2 control groups were 0.67 and 0.69, respectively）. Conclusions The anti-ODNs targeting the MSMEG_6401 gene could inhibit the growth of Mycobacterium smegmatls to some extent, and it could be supposed that the anti-ONDs might inhibit Mycobacterium tuberculosis. Rv3806c gene itself and its encoding protein might be a good therapeutic target for new tuberculosis drug development.

关 键 词：寡脱氧核糖核苷酸类 反义 分枝杆菌 耻垢 分枝杆菌 结核 

分 类 号：R686[医药卫生—骨科学]