长滩军团菌临床分离株与环境分离株全菌蛋白的比较与分析

Comparison and analysis of the total expressed protein of clinical and environmental isolates of Legionella longbeachae

目的采用蛋白质组学方法,对长滩军团菌血清1型临床分离株(LL-1)与环境分离株(CD-1)全菌蛋白进行比较和分析,探索军团菌临床株与环境分离株是否存在蛋白表达差异,为进一步认识、研究军团菌提供依据。方法培养、收集CD-1与LL-1菌株,用超声兼尿素-3-[(3-胆酰胺丙基)-乙二胺]-1丙磺酸(CHAPS)-二硫苏糖醇(DTT)裂解法提取全菌蛋白,SDS-PAGE及Western blot对二者全菌蛋白进行初步比较,确定二者全菌蛋白表达存在差异后,采用双向电泳(2-DE)联合MALDI-TOF-TOF质谱深入研究,并对质谱鉴定的差异点进行Western blot验证。结果初步SDS-PAGE及Western blot试验表明,CD-1与LL-1存在蛋白表达差异。进一步的2-DE研究结果共获得142个蛋白。CD-1与LL-1相比,18个蛋白点表达上调、19个蛋白表达下调。对其中显著差异的四个点进行质谱分析、Mascot软件查询、蛋白数据库搜库后证实CD-1表达下调的为热休克蛋白70(Chaperone protein Dna K)和30 s核糖体蛋白S1(30S ribosomal protein S1);CD-1表达上调的蛋白为假想蛋白TP04_0359(hypothetical protein TP04_0359)和一个未知蛋白。对进行质谱分析的四个蛋白点进行Western blot验证确实是军团菌蛋白。结论 LL-1与CD-1蛋白表达存在差异,此试验为进一步研究军团菌毒力和致病及疫苗研制奠定了基础。

Objective Through proteomic methods to compare and analyze the total expressed proteins of clinical and environmental isolates of Legionella longbeachae sgl, and explore the differentially expressed protein between them. And the aim is to lay the foundation for further understanding and study on Legionella spp. Methods The total expressed protein from the strains of CD- 1 and LL- 1 was extracted by Sonication-Urea-3- [ - （ 3-cholamidopropyl ） -dimethylammonio ] - 1-propanesulfonate （CHAPS）-DTI＂ method, and preliminarily compared through SDS-PAGE and Western blot. After the differentially expressed proteins were found, 2-dimensionnal electrophoresis（2-DE） and MALDI-TOF-TOF were employed to obtain more information of the protein. And then Western blot was adopted to verify differentially expressed protein spots which identified by mass spectrometry. Results The preliminary data from SDS-PAGE and Western blot showed that the differentially expressed proteins exist between CD-1 and LL-1. The 2-DE revealed 142 protein spots. Compared with LL-1 ,CD-1 has 18 protein spots up-regulated and 19 down-regulated. Among the differentially expressed protein spots, 4 significant difference spots of them were identified as Heat shock protein 70, 30S ribosomal protein $1, hypothetical protein TP04_0359 and the unknown protein by mass spectrometry, Mascot inquiring and database searching in CD-1. And the first two proteins were down-regulated while the following two were up-regulated. The results of Western blot demonstrated that the 4 differentially expressed protein spots were belong to Legionella. Conclusion There are differences between the total expressed protein of CD-1 and LL-1.The study may be helpful for virulence and pathogenic an alysis of Legionella, and stand as a valuable resource for vaccine development against Legionella.