【摘要】 利用板状聚丙烯酰胺凝胶电泳 (PAGE)方法 ,建立了一步分离纯化基因工程表达蛋白质的新技术。在构建的人绒毛膜促性腺激素嵌合肽 (CP1 2 )表达率约为 1 %的情况下 ,借助分析制备两用型板状PAGE装置 ,通过下行和上行两次电泳以及用透析膜隔离形成收集槽这两步改进 ,从包涵体抽提液中一步纯化了目的表达产物。结果表明 ,一次上样总蛋白质量为 3～ 4mg的PAGE ,可获得 5 0～ 1 0 0 μgCP1 2蛋白 ,其相对均一性达到 95 %。研究提示此改良的制备性PAGE新技术 ,是一种分离纯化低分子量目的蛋白质的好方法更多还原

【Abstract】 In recent years, development of chimeric peptide ( CP ) immunogens is a trend in the vaccinological field. The CPs contain a B cell epitope(s) of target antigen and a “promiscuous” self or foreign T cell epitope(s). However, such constructed CPs were all expressed in prokaryotic or eukaryotic systems at lower levels. To purify the human chorionic gonadotropin (hCG) CP12 expressed in E.coli at the level of about 1% of the total cell proteins, an improved method of preparative gel polyacrylamide gel electrophoresis (PAGE) was developed. The important improvement to routine preparative PAGE involves: (1) running reversed electrophoresis by rearranging the gel carrying plate when the bromophenol blue band arrived at 1—1.5 centimeter from the bottom of the gel; (2) making a collecting trough between the gel and a dialytic membrane that was used to isolate the upper tank buffer. About 8 fractions were collected at regular intervals of 15 minutes after bromophenol blue running out of gel. And then 0.2 ml was taken from each fraction and the protein was precipitated by sequentially adding trichloroacetic acid and acetone. Each sample was dissolved in 20 μl sample buffer and analyzed and identified by SDS PAGE and Western blotting. As a result, the hCG CP12 expression product with 95% relative homogeneity was harvested at a 50—100 μg level after a single step purification of this preparative PAGE, with respect to the sample which contained 3—4 mg of cell proteins.更多还原