论文部分内容阅读
目的研究胃癌组织中野生型和突变型DNA聚合酶β在DNA修复过程中的作用,为进一步探讨肿瘤的病因发病学奠定基础。方法采用聚合酶链反应(PCR)和分子克隆技术,以野生型和突变型DNA聚合酶β基因为模板,扩增后构建pGEM-T-polβ克隆载体,经PCR筛选,亚克隆于pET28a(+)表达载体中,并转化至大肠杆菌BL21(DE3)感受态细菌,经诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达后,通过镍柱亲和层析法纯化蛋白。设计3条单链DNA引物,退火合成含有单碱基缺失的DNA底物,与纯化蛋白共做碱基切除修复(BER)实验,最后琼脂糖凝胶电泳鉴定。结果成功构建了pET28a(+-)polβ重组表达载体;在BER实验中,野生型DNA聚合酶β能够在DNA底物的酶切位点处将其切开,突变型DNA聚合酶β不能或部分将其切开。结论野生型DNA聚合酶β能够修复单碱基缺失的DNA底物,而突变型DNA聚合酶β在碱基缺失修复过程中的作用丧失或减弱。
Objective To study the role of wild-type and mutant DNA polymerase-β in DNA repair in gastric cancer tissues, and lay a foundation for further exploration of the etiology and pathogenesis of gastric cancer. Methods Polymerase chain reaction (PCR) and molecular cloning techniques were used to amplify the pGEM-T-polβ cloning vector with wild-type and mutant DNA polymerase β as template. The PCR products were subcloned into pET28a (+ ) Expression vector and transformed into Escherichia coli BL21 (DE3) competent cells. After induced by the inducer IPTG, the recombinant protein was purified by nickel column affinity chromatography protein. Three single-stranded DNA primers were designed and annealed to synthesize a DNA substrate with a single base deletion. A base excision repair (BER) experiment was performed with the purified protein, and the final agarose gel electrophoresis was performed. Results The recombinant plasmid pET28a (+ -) polβ was successfully constructed. In the experiment of BER, the wild type DNA polymerase β could cleave it at the restriction site of DNA substrate. The mutant DNA polymerase β could not or partially Cut it. Conclusion Wild-type DNA polymerase β can repair DNA substrates with single-base deletion, and the effect of mutant DNA polymerase β in the process of base-deletion repair is lost or weakened.