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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。
Objective To explore the relationship between the structure and function of tumor suppressor gene OVCA1 and to elucidate the mechanism of its function in tumorigenesis. In this study, OVCA1A34D mutant was constructed and the effect of site mutation on its half-life was explored. Methods The recombinant plasmids of GFP-tagged-OVCA1A34D mutant were constructed by molecular cloning using the plasmid containing the full-length sequence of human OVCA1 gene prepared and preserved in our laboratory as a template and confirmed by sequencing. The mutant plasmids were transfected into cultured cells by liposome method to observe the expression of OVCA1A34D mutant protein in the cells. The effects of mutation of OVCA1A34D gene on the bio-half life of protein were determined by cycloheximide inhibition of protein synthesis. Results The GFP-tagged-OVCA1A34D mutant recombinant plasmid was successfully constructed and successfully expressed in the cells. The half-life of the OVCA1A34D mutant protein was significantly longer compared to the wild-type. Conclusion OVCA1A34D site mutation can lead to the extension of its biological half-life.