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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。
The GST fusion protein expression system was combined with protein truncation detection (PTT) to detect the frameshift mutation of Lis1 in HCC tissues. That is, the Lis1 gene amplified by RT PCR from the liver cancer tissue was cloned into the GST fusion protein expression vector pGEX 1 and expressed in E. coli DH5α. The truncated GST Lis1 fusion protein was found by SDS PAGE electrophoresis in a size of 33 kD, while the full-length fusion protein should be 71 kD in size. Sequencing verified that the Lis1 gene that produces the truncated protein was found to have an additional A between the 16th and 16th nucleotides, resulting in a frameshift mutation in the reading frame leading to an early termination of the translation, resulting in a truncated Protein product. Lis1 gene frameshift mutation in this frame is associated with liver cancer, pending further verification. However, the experimental results show that the improved PTT method is indeed a fast and effective method to detect frameshift mutations.