CAJ | 학술논문

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3 h 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2 d,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-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。
목적：용동원중조방법구건치구분지간균 MC2155단백매체보조인자 A(proteasome accessory factor A,PafA)기인고제균주,초보탐구 PafA 기인적결실대치구분지간균적생장활력급약물민감성적영향。방법대치구분지간균기인조중 PafA 량측서렬분별진행확증,련접재체급목적편단구건자살재체,전격전입치구분지간균중,여기인조중적 PafA 진행동원교환,통과람백반사선출기인고제균주。대고제균주여야생균주매격3 h 측정기정상조건、염양조건하급가불동농도이연정、리복평화을알정순조건하균액적흡광도(A580)치,비교기생장활력,련속2 d,회제생장곡선。결과통과동원중조방법성공득도료치구분지간균적 PafA 기인고제주,불동약물농도급생장조건하량자적생장곡선정“S”형,량자지간적생장속도미견명현차별。결론 PafA 수연시원핵세포류범소-단백매체계통중적중요조성부분,단타적결실병미인기현저적균주생장활력화약물민감성적변화,제시 PafA 적주요공능가능불재우차,균주령외환유대상적도경,PafA 기인공능병불시영향치구분지간균생장급약물작용효과적유일인소,대타적공능연구수요경가심입。
Objective To establish Mycobacterium smegmatis MC2 1 55 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 1 55 genome were amplificated and inserted to a vector to construct a suicide vector.Gene exchange took place within the MC2 1 55 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 A5 80 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.