空肠弯曲菌的双向电泳系统可重复性评价

The stability and reproducibility of two-dimensional gel electrophoresis for Campylobacter jejuni

目的:对空肠弯曲菌(Cj)的双向电泳(2DE)系统可重复性和稳定性进行评价,为空肠弯曲菌的蛋白质组学研究奠定基础。方法:选取一株空肠弯曲菌,培养48小时后制备全菌蛋白,进行双向电泳。比较不同上样量和不同批次对实验结果的影响。结果:不同批次和不同上样量的胶进行比较,蛋白质的匹配率均可达到90.89%;未匹配蛋白主要是一些低丰度蛋白;增加上样量可增加低丰度蛋白的检出,但可产生蛋白质的共迁移,因此选择合适的上样量很重要;不同批次蛋白质点在等电点方向的位置偏移为0.62±0.37mm,在SDS-PAGE方向的位置偏差为0.72±0.51mm,相对含量的相关系数为0.973。结论:表明空肠弯曲菌2DE的系统具有很好的稳定性及很高的可重复性,符合蛋白质组学的要求,实验结果具有可比性。

Objective： To evaluate the reproducibility of two-dimensional gel electrophoresis（2DE） for Campylobacter jejuni （C. jejuni） and to lay the foundation for the proteomic study of C. jejuni. Methods： A C. jejuni strain was selected and cultured for 48 hours. The whole-cell protein sample was prepared and separated by 2DE. The influence of different sample loadings and different times on the protein detection was observed. Results： The percentage of matched proteins between gels performed at different times but in the same sample loading and between gels performed at the same time but in different sample loading both could reach 90.89% The unmatched proteins were mainly lower abundance proteins; The detection of lower abundance proteins could be increased by increasing the sample loading, but the comigration of proteins occured, so it is important to choose the optimum sample loading; The positional variability of spots in gels performed at different times was 0.62 ±0.37mm in isoelectric focusing dimension and 0. 72±0. 51mm in SDS-PAGE dimension. The correlation coefficient of protein quantitation was 0. 973. Conclusion： The results show that the 2DE system for C. jejuni which is stable and reproducible can meet the needs of the proteomics.