【摘要】 目的为探索抑癌基因OVCA1结构与功能的关系,阐明其在肿瘤发生发展中的作用机制,本研究构建OVCA1A34D突变体并探讨该位点突变对其生物半衰期的影响。方法以本实验室制备并保存的含有人OVCA1基因全长序列的质粒为模板,采用分子克隆方法构建了GFP-tagged-OVCA1A34D突变体重组质粒,并经测序证实。应用脂质体法将突变体质粒转染入体外培养细胞,观察OVCA1A34D突变体蛋白在细胞中的表达。采用蛋白合成抑制剂放线菌酮抑制蛋白合成后检测OVCA1A34D基因突变对其蛋白生物半衰期的影响。结果成功构建了GFP-tagged-OVCA1A34D突变体重组质粒,并在细胞中成功表达。OVCA1A34D突变体蛋白的半衰期与野生体相比明显延长。结论 OVCA1A34D位点突变可导致其生物半衰期延长。更多还原

【Abstract】 Objective To explore the relationship between the structure and function of tumor suppressor OVCA1,in order to clarify the mechanism of OVCA1 in tumorigenesis and progression.Methods The GFP-tagged-OVCA1A34 Dmutant was built by gene cloning and was confirmed by sequencing.The mutant was transfected into cells with liposome assay.The expression of OVCA1A34 Dmutant protein was detected and the half-life of the protein was measured by inhibiting protein synthesis with cycloheximide(CHX).Results GFP-tagged-OVCA1 A34 D was established successfully and was expressed in cells.The half-life of OVCA1A34 Dmutant protein was longer compared with that of wild type OVCA1 protein.Conclusion OVCA1A34 Dmutation extends the protein′s half-life.更多还原