【摘要】 将GST融合蛋白表达系统与蛋白质截短检测法 (PTT)法相结合 ,检测Lis1基因在肝癌组织中的阅读框移码突变。即从肝癌组织中通过RT PCR扩增的Lis1基因克隆入GST融合蛋白表达载体pGEX 1,并于大肠杆菌DH5α中进行表达。通过SDS PAGE电泳发现肝癌组织中有截短的GST Lis1融合蛋白 ,大小为 33kD ,而全长融合蛋白大小应为 71kD。经测序验证 ,发现产生截短蛋白的Lis1基因在第 16 3位和第 16 4位核苷酸之间多了一个A ,致使阅读框发生移码突变 ,导致翻译提早终止 ,从而产生截短的蛋白质产物。Lis1基因的这种阅读框移码突变是否与肝癌相关 ,有待进一步验证。但实验结果证明改进后的类PTT法用于检测基因阅读框移码突变确实是一种快速有效的方法。更多还原

【Abstract】 GST fusion protein expression system combined with protein truncation test(PTT) protocol was used to detect gene frame shift mutation. The RT PCR products of Lis 1 genes from hepatocarcinoma samples were respectively cloned into a GST fusion protein expression vector pGEX 1, then expressed in E.coli . The results showed a truncated 33 kD fusion protein in SDS PAGE, although the full translated product of Lis 1 gene should be of 71 kD. Sequencing revealed insertion of an A residue, causing the premature termination of translation, between the 163th and 164th nucleotide of Lis 1 gene. This improved PTT assay was proved to be a fast and effective way in detecting gene frame shift mutation.更多还原