人血小板膜糖蛋白ITGB3基因终止突变c.1476G＞A的体外表达研究

摘要

Objective To explore the function of a novel nonsense mutation c.1476G＞A of ITGB3 gene using an in vitro expression system.Methods An eukaryotic expression vector containing ITGB3 c.1476G＞A eDNA was generated by site-directed mutagenesis and transformed into E.coli.Plasmid DNA was extracted and sequenced to confirm the target mutations.Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method,and the stable expression cells were harvested by G418 screening.The ITGB3 gene mRNA transcription and GP Ⅲ a expression level in CHO cells were detected with real-time quantitative PCR,Western blotting and flow cytometry,respectively.Results The eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G＞A mutant were successfully constructed.CHO cells with stable expression were obtained after transfection and screening.Compared with the wild-type transfected cells,the amount of CD61 antigen expression was 37 ％ and mRNA transcription level was only 6％ in the mutant-transfected cells.Full length GPⅢ a protein was found only in the stably wild-type-transfected cells,but not in mutant-transfected cells by Western blotting analysis.Conclusion The ITGB3 c.1476G＞A mutation can decrease the transcription level and further affect GPⅢ a synthesis and CD61 antigen expression.%目的 对ITGB3基因新的终止突变c.1476G＞A进行体外表达研究,以阐明其功能.方法 应用体外定点诱变技术构建突变型ITGB3 c.1476G＞A cDNA的真核表达载体,转化感受态细胞,提取质粒后测序验证.采用非脂质体介导重组质粒转染中国仓鼠卵巢癌细胞株(Chinese hamster ovarian cancer,CHO)细胞,经G418筛选获取稳定表达细胞株.采用实时荧光定量PCR、Western印迹和流式细胞术分别检测CHO稳定表达株ITGB3基因的mRNA表达和血小板膜糖蛋白Ⅲa(glycoproteinⅢa,GPⅢa)糖蛋白水平.结果 成功构建野生型和突变型ITGB3 cDNA的真核表达载体,转染筛选后分别获得了稳定表达的CHO细胞株.突变型细胞株中CD61抗原表达量仅为野生型的37％,而ITGB3 mRNA转录水平为野生型的6％.Western印迹结果显示,野生型细胞株稳定表达完整的GPⅢa蛋白,而突变型CHO细胞株无完整的GPⅢa蛋白表达.结论ITGB3 c.1476G＞A终止突变可导致ITGB3基因的转录水平降低,影响GPⅢa蛋白合成及CD61抗原的表达水平.