中药天花粉蛋白的定点突变及突变体的活性检测

Site-mutation and the Activity Assay of Mutants of Trichosanthin, A Chinese Herb Medicine

选择天花粉蛋白 (Trichosanthin ,TCS)分子中两个可能的抗原决定簇位点 :Tyr81 Phe82 Phe83和Tyr94 Val95 Phe96进行了定点突变 ,将它们分别替换成Ala81 Gly82 Ser83和Ala94 Val95 Gly96。这两个突变体蛋白分别在大肠杆菌中成功地表达进行纯化 ,纯化后的突变体蛋白经初步检测发现其活性与野生型蛋白的活性几乎相同 ,而YFF81 83AGS突变体的免疫原性有所降低。

Site directed mutation was performed to investigate the key amino acids for immunogenicity of Trichosanthin. Two possible antigenic sites, YFF81 83 and YVF94 96, were replaced by Ala81 Gly82 Ser83 and Ala94 Val95 Gly96, respectively, to obtain two mutants, YFF81 83AGS TCS and YVF94 96AVG TCS. These two mutants were expressed in E. coli DH5α and purified. The DNase activities of the mutants were examined. It was shown that there was no significant difference between the two mutants of TCS and the natural TCS. While the mutant YFF81 83AGS appeared a lowing immunogencity.