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本研究探讨35C>T和682A>G突变点对α-1,2岩藻糖基转移酶(FUT1)表达活性的影响。PCR扩增FUT1全长编码序列DNA片段,通过TOPOTA技术连接至表达载体pcDNA3.1/V5-His,获取目的重组质粒。采用脂质体转染方法将重组质粒转染COS-7细胞,经G418筛选培养后,利用流式细胞术分析细胞表面H抗原,实时荧光PCR检测fut1 mRNA表达情况。结果表明:成功获取了35T、682G+35T和682A+35C重组质粒。转染2天后H抗原在COS-7细胞膜上表达,第4天达最高峰。与野生型(682A+35C)转染的细胞相比,第4天重组质粒(682G+35T)转染细胞H抗原的表达量仅为野生型的13.3%,而重组质粒35T转染细胞为野生型的52.7%。转染后fut1mRNA水平随着时间延长而逐渐递减,在第1天重组质粒(682G+35T)转染细胞fut1 mRNA水平仅为野生型(682A+35C)的14%。结论:682A>G突变明显降低α-1,2岩藻糖基转移酶活性,而35C>T引起H抗原的表达部分下降。
This study was designed to investigate the effect of 35C> T and 682A> G mutation on the expression of FUT1 in α-1,2-fucosyltransferase. The FUT1 full-length coding sequence DNA fragment was amplified by PCR and linked to the expression vector pcDNA3.1 / V5-His by TOPOTA technology to obtain the desired recombinant plasmid. The recombinant plasmids were transfected into COS-7 cells by lipofection method. After G418 selection and culture, the cell surface H antigen was analyzed by flow cytometry. The expression of fut1 mRNA was detected by real-time fluorescence PCR. The results showed that 35T, 682G + 35T and 682A + 35C recombinant plasmids were successfully obtained. After 2 days of transfection, H antigen was expressed on the membrane of COS-7 cells, reaching its peak on the 4th day. Compared with the wild-type (682A + 35C) transfected cells, the expression level of H antigen in the transfected cells on day 4 was only 13.3% of the wild type, while that of the recombinant plasmid 35T was wild Type 52.7%. After transfected fut1mRNA levels gradually decreased over time, in the first day recombinant plasmid (682G +35 T) transfected cells fut1 mRNA level only wild-type (682A +35 C) of 14%. CONCLUSION: The 682A> G mutation significantly decreased the activity of α-1,2 fucosyltransferase, whereas the 35C> T induced a partial decrease of H antigen expression.