广州管圆线虫Ⅴ期幼虫蛋白质组双向凝胶电泳技术的建立和优化

Establishment and condition optimization of two-dimensional gel electrophoresis on proteomic in the study of Angiostrongylus cantonensislarvaeⅤ

目的优化双向凝胶电泳的条件,建立适合广州管圆线虫Ⅴ期幼虫的最适二维电泳模型。方法经研磨、反复冻融提取广州管圆线虫Ⅴ期幼虫蛋白质,测定其浓度后分别用pH3～10和pH4～7的固相胶条进行双向凝胶电泳,同时对双向电泳的条件进行调节和优化,银染后进行凝胶图像比较。结果采用pH3～10的胶条分离蛋白时,蛋白质点分布较集中,各蛋白点间间距较小;pH4～7的胶条分辨率较高,蛋白分布均一,获得719个蛋白点,各蛋白点间距相对较大,避免了高丰度掩盖低丰度蛋白点,在相同的pH范围内,检测到的蛋白点更多,灵敏度更高。结论建立和优化适合广州管圆线虫的二维电泳模型,其重复性好、分辨率高,为广州管圆线虫的蛋白质组学研究奠定了基础。

To establish and optimize two-dimensional gel electrophoresis （2-DE） models for proteomic analysis of A. cantonensis larvaeV , the protein of larvae V of A. cantonensis was extracted by grinding and repeated freezing and thawing, then 2-DE was carried out respectively with the strip of pH3-10 and pH4-7 after measuring the concentration. The gel electro- phoresis image was compared after silver staining. The results indicated that the protein spots were more concentrated, and the space among spots were smaller with IPG strip of pH3-10. Then we obtained high-resolute, uniform distributed protein spots, 719 protein spots with IPG strip of pH4-7, and the space among spots were relatively large, avoiding to protein spots of high a- bundance cover the low abundance, it has higher sensitivity. As a result, establishment and optimization of 2-DE models for A. cantonensis was successfully conducted, and 2-DE gel with high resolution was obtained, which might provide foundation for the proteomic analysis of A. cantonensis.