蛋白质双向电泳、印迹转移及蛋白质合成无细胞体系技术在小鼠腹水细胞EF-3研究中的应用

本文应用近年发展起来的生化技术――蛋白质双向电泳（其第一向为等电聚焦，第二向为SDS凝胶电泳），将小鼠腹水细胞核糖体蛋进行了指纹分离。并利用蛋白质印迹转移（Western blotting），将转移后的硝酸纤维膜与交联了碱性磷酸酶的第二抗体和抗酵母EF-3抗体反应，证实该核糖体蛋白含有EF-3同源片段，进而制备了蛋白质合成无细胞体系。通过测定PolyU指导下3 H-phe掺入活力的免疫失活实验，初步证实此同源片段是小鼠腹水细胞蛋白质合成所必需。 The ribosomal proteins of H22a cell,were separated with the method of two-D gel electrophoresis (the first dimention is isoelectric focus and the second is SDS PAGE).Then the Western blotting was used,the transferred nitrocellulose sheet was treated with antiyeast EF-3 antibody and the second antibody bonded with alklinephosphoesterase.The result shows that the ribosomal proteins have a homologous fragment to yeast EF-3 factor.The cell-free system of protein synthesis was also established.By determing the activity of polyU direeted 3H-phe intervention in immunodeactivitive experiment,it is primaril confirmed that this fragment is the esscntial for the protein biosynthesis in H22a cell.

The Application of the Methods of Two Dimentional Gel Electrophoresis,Western Blotting and Cell-free System of Protein Synthesis in the Research in EF-3 Factor of H22a Cell