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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 To compare and analyze the total bacterial proteins of Legionella pneumophila serotype 1 clinical isolates (LL-1) and environmental isolates (CD-1) using proteomics methods and to explore the existence of Legionella isolates Protein expression differences, in order to further understand and study Legionella to provide the basis. Methods The CD-1 and LL-1 strains were collected and cultured with ultrasound and urea-DTT (CHAPS) -Dithiothreitol (DTT) ) Were separated by SDS-PAGE and Western blot. The results showed that there was a difference in the expression of whole-cell proteins between the two strains. The two-dimensional electrophoresis (2-DE) and MALDI-TOF-TOF mass spectrometry In-depth study, and identification of the differences in mass spectrometry Western blot validation. Results The results of SDS-PAGE and Western blot showed that the protein expression of CD-1 and LL-1 were different. A total of 142 proteins were obtained from further 2-DE studies. Compared with LL-1, CD-1 showed up-regulated expression of 18 protein spots and down-regulated expression of 19 proteins. Mass spectrometry was performed on the four spots with significant difference. Mascot software was searched and the database of protein databases was searched to confirm that the expression of CD-1 was down-regulated by Chaperone protein Dna K and 30S ribosomal protein S1 S1). The up-regulated protein of CD-1 was hypothetical protein TP04_0359 (hypothetical protein TP04_0359) and an unknown protein. Western blotting of the four protein spots for mass spectrometry proved to be Legionella proteins. Conclusion There is a difference in the expression of LL-1 and CD-1 proteins. This test lays a foundation for further study on the virulence and pathogenesis of Legionella and the development of vaccine.