红芪多糖3作用小鼠胸腺组织蛋白双向电泳技术的建立

Establishment of Two-dimensional Gel Electrophoresis Techniques for Proteins from Mouse Thymus Tissue Treated with Third Hedysarum Polybotys Saccharide

目的建立和优化红芪多糖3(HPS-3)作用后小鼠胸腺组织蛋白质组双向电泳技术方法。方法采用标准裂解法提取HPS-3 100 mg/(kg.d)灌胃14 d小鼠的胸腺组织总蛋白,后续进行三氯乙酸(TCA)-丙酮沉淀法和去脂蛋白纯化法获取胸腺组织蛋白,以固相pH梯度胶条进行双向凝胶电泳,并对等电聚焦程序进行改良和优化,同时将去脂蛋白纯化法运用于A549细胞蛋白的纯化并进行双向电泳,银染色后进行凝胶图像比较。结果去脂蛋白纯化法不仅能减少蛋白降解,而且增加蛋白的溶解性,因此去脂蛋白纯化法所得蛋白的双向电泳图谱显示更好的溶解性和重复性,此外在双向凝胶电泳(2-DE)时,聚焦电压在传统聚焦条件上进行优化,使横向和纵向拖尾有明显改善,成功建立了背景清晰,蛋白分离良好、分辨率较高的胸腺组织蛋白的双向电泳体系。去脂蛋白纯化法同样适用于A549细胞蛋白提取纯化。结论建立了HPS-3作用后小鼠胸腺组织蛋白质组提取纯化的方法,采用去脂蛋白纯化法可以更有效地提取胸腺组织蛋白。利用优化后的实验方法,获得了HPS-3作用后小鼠胸腺组织蛋白2-DE图谱,为后续研究打下基础。

Objective To establish and optimize the technique of two-dimensional gel electrophoresis（2-DE） for proteins in mouse thymus tissue treated with the third polysaccharide from Radix Hedysari（HPS-3）. Methods Thymus totally proteins from the mice who had been given HPS-3 in 100 mg/（kg·d） for fourteen days were extracted using standard tissue lysis method,then respectively using trichloroacetic acid（TCA）-acetone precipitation method and delipidation and protein precipitation method were obtained.In the meantime,the delipidation and protein precipitation method was utilized to precipitate the protein from A549 cell. The protein was separated using two-dimensional gel electrophoresis with immobilized pH gradient condition of isoelectric focusing（IEF）were also adjusted and optimized,then the gels were stained by beyotime fast sliver stain kit and compared the digitized images of 2-DE using image scanner. Results Delipidation and protein precipitation method not only decreased protein decomposition,but also increased protein solubility,therefore the two-dimensional electrophoresis spectrum was obtained with higher resolution and better reproducibility.Besides,the focus voltage for gel electrophoresis was optimized on the traditional focus conditions,and a two-dimensional electrophoresis system with a clear background and good protein separation was successfully established for thymus tissue.The optimized focusing condition made horizontal and vertical tail improved obviously,and a high-resolution two-dimensional electrophoresis map with clearer protein spots and more complete separation obtained easily,the Delipidation and protein precipitation method was also suitable for the protein of A549. Conclusion The method of extracting thymus tissue protein was established.The protein spots in 2-DE map can significantly increase using chemical delipidation protein purification.In addition,by using the optimized method described above,satisfactory 2-DE maps of thymus tissue can be obtained,which lays a foundation for the further study of the protein of thymus tissue treated with HPS-3.