耻垢分枝杆菌蛋白酶体辅助因子A的敲除和功能探讨

Effect of proteasome accessory factor A deficient to growth and drug sensitivity of mycobacterium

目的用同源重组方法构建耻垢分枝杆菌MC2155蛋白酶体辅助因子A （ proteasome accessory factor A,PafA）基因敲除菌株，初步探究PafA基因的缺失对耻垢分枝杆菌的生长活力及药物敏感性的影响。方法对耻垢分枝杆菌基因组中PafA两侧序列分别进行扩增，连接载体及目的片段构建自杀载体，电击转入耻垢分枝杆菌中，与基因组中的PafA进行同源交换，通过蓝白斑筛选出基因敲除菌株。对敲除菌株与野生菌株每隔3h测定其正常条件、厌氧条件下及加不同浓度异烟肼、利福平和乙胺丁醇条件下菌液的吸光度（A580）值，比较其生长活力，连续2d，绘制生长曲线。结果通过同源重组方法成功得到了耻垢分枝杆菌的PafA基因敲除株，不同药物浓度及生长条件下两者的生长曲线呈“s”形，两者之间的生长速度未见明显差别。结论PafA虽然是原核细胞类泛素一蛋白酶体系统中的重要组成部分，但它的缺失并未引起显著的菌株生长活力和药物敏感性的变化，提示PafA的主要功能可能不在于此，菌株另外还有代偿的途径，PafA基因功能并不是影响耻垢分枝杆菌生长及药物作用效果的唯一因素，对它的功能研究需要更加深入。

Objective To establish Mycobacterium smegmatis MC2 155 proteasome accessory factor A （PafA） gene knockout strain by homologous recombination and study the effect of PafA deficient on growth and drug sensitivity of mycobacterium. Methods The two sides fragments of PafA in Mycobacterium smegmatis MC2 155 genome were amplificated and inserted to a vector to construct a suicide vector. Gene exchange took place within the MC2 155 after the suicide delivery vectors was transformed into the strain. The strain of Mycobacterium smegmatis with PafA gene knockout was selected and the growth curve of wild type and PafA knockout type were studied under different concentration of isoniazid, rifampicin, ethambutol in normal condition and anaerobic condition, the value of As8o per three hours was detected for two days. Results The gene PafA knockout strain was successifully generated and a ＂S＂shaped growth curve was detected. However, there was no significant difference in the growth rates between the wild type and PafA knockout strains. Conclusions Although the PafA is an important part in prokaryotic ubiquitin-like protein-proteasome system,it has no significant effect on the vability and sensivity to the drugs, so the protein PafA may have other roles and the strain has a compensatory pathway. PafA is not the only factor to affect the growth and sensitivity to the drugs of mycobacterium smegmatis,and we should do more to find the functions.