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【目的】通过分离海分枝杆菌野生株和mkl突变株的全菌蛋白,并进行差异蛋白质组分析,以期为探索分枝杆菌重要毒力基因mkl的功能提供新思路。【方法】以海分枝杆菌野生株和mkl突变株为研究材料,提取全菌蛋白,i TRAQ试剂标记后进行质谱鉴定和定量分析,并利用Uni Prot数据库对差异蛋白进行生物信息学分析。【结果】共鉴定出在野生株和mkl突变株中差异表达蛋白566个,其中在突变株中上调表达蛋白232个(比值≥1.4),下调表达蛋白334个(比值≤0.7)。生物信息学预测这些蛋白主要参与细菌脂质代谢、细胞壁和细胞进程、中间代谢、呼吸作用等生物学功能。其中Des A3下调最显著,其功能为脂肪酸去饱和酶,与油酸合成相关,进一步验证发现mkl突变株在不含油酸的固体培养基中生长受限,提示mkl可能在油酸的生物合成通路中发挥功能。【结论】通过i TRAQ分析了海分枝杆菌mkl突变株和野生株的差异表达蛋白谱,发现可能影响分枝杆菌油酸、脂质等合成代谢通路,为进一步研究mkl基因在分枝杆菌致病中发挥作用的相关机制奠定了基础。
【Objective】 The purpose of this study is to provide new ideas for exploring the function of mkl, an important virulence gene of Mycobacterium, by isolating the whole mycoprotein of Mycobacterium maritima and mkl mutant and analyzing the differential proteome. 【Method】 Mycobacterium maritima and mkl mutants were used as materials to extract the whole-cell protein and labeled with i TRAQ reagent, then identified by mass spectrometry and quantified. Uni Prot database was used to analyze the differential proteins by bioinformatics analysis. 【Result】 A total of 566 differentially expressed proteins were identified in wild and mkl mutants, of which 232 were up-regulated (odds ratio ≥1.4) and 334 were down-regulated (odds ratio ≤0.7). Bioinformatics predicts that these proteins are mainly involved in the biological functions of bacterial lipid metabolism, cell wall and cell processes, intermediate metabolism, respiration and so on. Among them, Des A3 was most significantly down-regulated and its function was fatty acid desaturase, which was related to the synthesis of oleic acid. Further confirmation showed that the growth of mkl mutant in oleic acid-free solid medium was limited, suggesting that mkl may be involved in the oleic acid biosynthesis pathway In the function. 【Conclusion】 The differential expression protein profiles of Mycobacterium maritima mkl mutant and wild strain were analyzed by i TRAQ and found that it may affect the metabolic pathway of oleic acid and lipid of mycobacterium. To further study the mkl gene induced in Mycobacterium The mechanisms that play a role in the disease lay the foundation.