CAJ | 학술논문

本研究利用 MycoMarT7噬菌体建立了海分枝杆菌随机突变库，鉴定获得1株转座子插在基因MMAR＿0994（mkl）上的突变株。用野生株和突变株分别感染斑马鱼成鱼，通过观察斑马鱼的存活情况，研究 mkl基因对海分枝杆菌毒力的影响；利用尾静脉注射感染孵出48 h后的斑马鱼幼鱼，于荧光显微镜下观察突变株及野生株在幼鱼体内的播散情况，以检测 mkl基因在细菌与宿主天然免疫相互作用中的功能；将野生株和突变株分别感染小鼠来源巨噬细胞，检测其在巨噬细胞内的增殖。此外，还检测了突变株对酸性环境的耐受情况。结果显示，mkl基因突变株感染斑马鱼成鱼后25 d内没有鱼死亡；在斑马鱼幼鱼感染模型和细胞感染模型中，突变株的增殖明显减弱；突变株对酸性环境压力更敏感。以上结果提示，mkl基因在海分枝杆菌抵抗宿主免疫及致病中发挥重要作用。
본연구이용 MycoMarT7서균체건립료해분지간균수궤돌변고，감정획득1주전좌자삽재기인MMAR＿0994（mkl）상적돌변주。용야생주화돌변주분별감염반마어성어，통과관찰반마어적존활정황，연구 mkl기인대해분지간균독력적영향；이용미정맥주사감염부출48 h후적반마어유어，우형광현미경하관찰돌변주급야생주재유어체내적파산정황，이검측 mkl기인재세균여숙주천연면역상호작용중적공능；장야생주화돌변주분별감염소서래원거서세포，검측기재거서세포내적증식。차외，환검측료돌변주대산성배경적내수정황。결과현시，mkl기인돌변주감염반마어성어후25 d내몰유어사망；재반마어유어감염모형화세포감염모형중，돌변주적증식명현감약；돌변주대산성배경압력경민감。이상결과제시，mkl기인재해분지간균저항숙주면역급치병중발휘중요작용。
A Mycobacterium marinum random mutant library using MycoMarT7 phage system was constructed .After screening ,a mutant whose transposon insertion site was in MMAR_0994 (mkl) gene was obtained .The adult zebrafish were infected with the wild‐type and the mutant strains respectively and the survival rates of the zebrafish were compared .The potential impact of the mutant on host innate immune system was tested by infecting the 48‐hour post‐fertilization zebrafish larvae using caudal vein injection . In vivo distribution of the bacteria was monitored by following green fluorescence in the larvae .The impact of the mutant on mouse macrophage cell line was tested .In addition ,the acid tolerance level was also checked . The results showed that mkl mutant displayed attenuated virulence in the zebrafish infection model .The attenuated phenotypes in the zebrafish larvae and mouse macrophage cell line were observed .The invasion rates of the mutant and the wild‐type were similar .The growth of the mutant was more sensitive to low pH . These results indicated that mkl may play a functional role in the interaction with host immune system .