Chemale Gustavo, van Rossum Arjan J, Jefferies James R, Barrett John, Brophy Peter M, Ferreira Henrique B, Zaha Arnaldo
Centro de Biotecnologia and Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, 91501-970 RS, Brazil.
Proteomics. 2003 Aug;3(8):1633-6. doi: 10.1002/pmic.200300487.
We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.
我们描述了用于二维电泳（2-DE）的细粒棘球绦虫囊尾蚴蛋白提取物的制备方法。原头节和棘球蚴液通过三氯乙酸（TCA）沉淀法制备，以去除非蛋白质污染物。与未处理的对照相比，TCA沉淀改善了原头节蛋白的2-DE凝胶图谱。对原头节蛋白提取物不溶性和可溶性部分的2-DE凝胶进行比较，结果表明，超声裂解后大多数蛋白质不溶。宿主血清蛋白，尤其是白蛋白和球蛋白，因其在该样本中的高含量，在棘球蚴液2-DE凝胶上导致水平条纹问题。即使制备了棘球蚴液寄生虫富集部分，大量的牛血清白蛋白和球蛋白仍使得通过2-DE难以检测到寄生虫特异性蛋白。尽管缺乏细粒棘球绦虫基因组测序或表达序列标签（EST）项目，但通过肽质量指纹图谱从原头节全蛋白2-DE凝胶中鉴定出15个突出的蛋白斑点还是有可能的。这些蛋白包括肌动蛋白、原肌球蛋白、副肌球蛋白、硫氧还蛋白还原酶、抗原P-29、亲环蛋白以及热休克蛋白hsp70和hsp20。这项工作表明，2-DE和PMF是鉴定棘球蚴液和原头节中蛋白质以及对来自不同宿主的囊肿或活动期与静止期囊肿进行比较分析的重要工具。
