二维薄层色谱法分析分枝杆菌脂质成分

Analysis of mycobacterial lipid components by thin layer chromatography

为探讨卡介苗巴氏德菌株(Bacillus Calmette-Guérin-pasteur,BCG-pasteur)和耻垢分枝杆菌(Mycobacterium smegma-tis,mc^2155)两种菌株的脂质成分,且通过比较脂质成分差异为研发新型牛结核病脂类疫苗佐剂提供试验基础。本文通过石油醚,氯仿,甲醇,丙酮等有机溶剂萃取BCG-pasteur和mc^2155两种菌株脂质,采用薄层色谱法分析其非极性脂质成分的差异。结果显示,BCG-pasteur中脂质成分富含三酰甘油,甲硫酚二角酰亚胺,PAT家族蛋白,甘油二酯,游离脂肪酸,游离分枝菌酸,葡萄糖单霉菌酸酯以及硫脂I类和硫脂III类等物质;mc^2155脂质成分除了硫脂I类,其他成分均有表达,但相对于BCG-pasteur其含量明显减少。本萃取方法简便稳定,适用于分枝杆菌的非极性脂质提取,为分枝杆菌脂质成分的分析提供基础;本试验通过薄层色谱法分析显示卡介苗和耻垢分枝杆菌细胞壁脂质成分差异明显,表明不同结核分枝杆菌的细胞壁脂质成分存在明显差异,从而可以引起不同的免疫反应,可为新型牛结核病疫苗提供方向。

In order to generate a new bovine tuberculosis vaccine,we focused on mycobacterial lipid component as target of the new vaccine.We first established a method to evaluate the difference of the mycobacterial lipid components base on thin layer chromatography(TLC).We extracted the lipids of BCG-pasteur and mc^2155 by organic solvents such as petroleum ether,chloroform,methanol and acetone.Two-dimensional thin layer chromatography was used to analyze and compare the lipid components content.The results showed highly expressed triacylglycerol,methylthiol di-imide,PAT family protein,diglyceride,free fatty acid,free mycolic acid,glucose monomylate and sulphur lipid class I,as well as sulphur lipids III and other substances in the lipid component of BCG-pasteur.Except for sulphur fat class I,other ingredients were expressed in mc^2155.However the lipid in mc^2155 was less than BCG-pasteur strain.In Summary,we have established a method to extract the total mycobacterial lipids,which is simple and stable,and is suitable for non-polar lipid extraction of mycobacterial.We also find that there isa significant difference of lipid components between mc2155 and BCG-pasteur,and the differences of lipids in different strains may provide a new path to generate new bovine tuberculosis vaccine and adjuvants in future study.