摘要目的 研究α-1,2岩藻糖基转移酶基因(α-1,2 fucosyltransferase,FUT1)682A＞G和547_552delAG突变对FUT1转录和表达蛋白功能活性的影响.方法 构建FUT1 682A＞G和FUT1 547_552delAG真核重组表达质粒,并转染COS7细胞进行稳定表达细胞株筛选.实时荧光定量PCR检测FUT1 mRNA表达量,高效液相色谱检测FUT1酶的活性.结果 获得稳定表达FUT1 682A＞G、FUT1547_552delAG和FUT1野生型基因的COS7细胞,FUT1 682A＞G和547_552delAG重组体mRNA含量分别为野生型的101.69％和102.79％.聚丙烯酰胺凝胶电泳和Western印迹检测显示FUT1 682A＞G重组体细胞表达相对分子质量约为46 000的目的蛋白片段,而FUT1 547_552delAG重组体细胞未见同分子量大小的目的蛋白片段.酶活性分析显示FUT1 682A＞G重组体蛋白酶活性为野生型的61.06％,而547_552delAG重组体蛋白完全失去酶活性.结论 FUT1 682A＞G和547_552delAG突变不影响FUT1mRNA转录,但不同FUT1突变点对酶活性影响存在明显差异.

abstractsObjective To explore the effect of α-1,2 fucosyltransferase (FUT1)gene 682A＞G and 547_ 552delAG mutations on the expression of FUT1 mRNA and activity of α-1,2 fucosyltransferase.Methods Recombinant expression vectors of FUT1 682A＞G and FUT1 547_552delAG were constructed and transfected into COS-7 cells for stable expression screening.Expression of FUT1 mRNA was determined using real-time quantitative PCR.The activity of FUT1 was measured with high-performance liquid chromatography.Results Stably transfected COS-7 cells with wild type FUT1,FUT1 682A＞G and FUT1 547_552delAG were respectively obtained.The FUT1 mRNA level of transfected cells with 682A＞G and 547_552delAG recombination vectors have measured 101.69 ％ and 102.79 ％ compared with that of wild type FUT1 transfected cells.A specific protein band with about 46 kD was confirmed in the 682A＞G transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6 × His Tag antibody.Similar protein was not identified in the 547_552delAG cells lysates.Enzymes activity of FUT1 682A＞G has measured 61.01％ compared with wild type FUT1 protein,whilst the activity of FUT1 547-557delAG was completely abolished.Conclusion FUT1 682A＞G and 547_552delAG mutations do not affect the transcript efficiency,although various mutations have different impact on the enzyme's activity.