CAJ | 학술논문

目的建立α-1,2岩藻糖基转移酶基因(α-1,2-fucosyltransferase gene,FUT1)293C＞T和658C＞T突变真核表达细胞,阐明FUT1突变引起H抗原减弱的机制.方法抽提类孟买型先证者基因组DNA扩增FUT1全长编码序列,扩增片段与真核表达载体pcDNA3.1连接构建重组表达质粒.采用脂质转染技术将重组质粒转染COS7细胞并进行稳定表达筛选.采用实时荧光定量PCR检测mRNA表达量,应用HPLC检测酶活性,采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)和Western印迹技术鉴定蛋白.结果构建了pcDNA3.1-FUT1野生型、pcDNA3.1- FUT1 293C＞T、pcDNA3.1-FUT1 658C＞T真核表达重组质粒,转染后通过G418筛选获得了稳定表达FUT1的COS7细胞.以野生型重组体转染细胞中FUT1 mRNA量作为对照,293C＞T和658C＞T重组体转染细胞FUT1 mRNA量分别为野生型的97.10％和104.74％.经SDS-PAGE电泳和Western印迹检测显示细胞表达相对分子质量约46000大小的目的蛋白片段,pcDNA3.1-FUT1野生型重组体转染细胞表达蛋白可催化相应的酶促反应,而pcDNA3.1-FUT1 293C＞T、pcDNA3.1- FUT1 658C＞T转染细胞蛋白完全失去酶活性.结论体外实验提示293C＞T和658C＞T突变并不影响FUT1 mRNA转录和蛋白生成,但表达蛋白的酶活性明显下降,从而导致H抗原生成减弱.
목적 건립α-1,2암조당기전이매기인(α-1,2-fucosyltransferase gene,FUT1)293C＞T화658C＞T돌변진핵표체세포,천명FUT1돌변인기H항원감약적궤제.방법 추제류맹매형선증자기인조DNA확증FUT1전장편마서렬,확증편단여진핵표체재체pcDNA3.1련접구건중조표체질립.채용지질전염기술장중조질립전염COS7세포병진행은정표체사선.채용실시형광정량PCR검측mRNA표체량,응용HPLC검측매활성,채용십이완기광산납취병희선알응효전영(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)화Western인적기술감정단백.결과 구건료pcDNA3.1-FUT1야생형、pcDNA3.1- FUT1 293C＞T、pcDNA3.1-FUT1 658C＞T진핵표체중조질립,전염후통과G418사선획득료은정표체FUT1적COS7세포.이야생형중조체전염세포중FUT1 mRNA량작위대조,293C＞T화658C＞T중조체전염세포FUT1 mRNA량분별위야생형적97.10％화104.74％.경SDS-PAGE전영화Western인적검측현시세포표체상대분자질량약46000대소적목적단백편단,pcDNA3.1-FUT1야생형중조체전염세포표체단백가최화상응적매촉반응,이pcDNA3.1-FUT1 293C＞T、pcDNA3.1- FUT1 658C＞T전염세포단백완전실거매활성.결론 체외실험제시293C＞T화658C＞T돌변병불영향FUT1 mRNA전록화단백생성,단표체단백적매활성명현하강,종이도치H항원생성감약.
Objective To establish the eukaryotic cell expression system for the α-1,2 fucosyltransferase gene ( FUT1 ) 293C＞T and 658C＞T mutations and explore the mechanism of FUT1 mutations resulting in the reduced expression of H antigen.Methods Genomic DNAs were extracted from individuals with para-Bombay phenotype and full coding region of FUT1 was amplified.The amplification fragments were ligated with pcDNA3.1 plasmid to construct the recombinant expression vectors. The recombinant plasmids were transfected into the COS-7 cells using lipofectamine transfection reagent and stable expression screening was performed. FUT1 mRNA expression was determined by real-time quantitative PCR. The activity of enzyme was measured by high performance liquid chromatography.Expressed protein was identified by SDS-PAGE and Western blotting. Results pcDNA3.1- FUT1 wildtype,pcDNA3.1- FUT1 293C ＞ T and pcDNA3.1- FUT1 658C ＞ T recombinant vectors were constructed,respectively.COS-7 cells with stable expression of FUT1 were obtained through recombinant plasmid transfection and screening with G418.The FUT1 mRNA level of transfected cells with 293C＞T and 658C＞T recombinant vectors reached 97.10％ and 104.74％ of the wildtype FUT1 transfected cell.A specific protein band with about 46000 was confirmed in the transfected cell lysates by SDS-PAGE electrophoresis and Western blotting with 6 × His Tag antibody.The wildtype FUT1 transfected cell lysates can catalyze the enzymatic reaction,while the enzyme activity of cell lysates from 293C＞T and 658C＞T were abolished.Conclusion The results suggested that the 293C＞T and 658C＞T mutations of FUT1 gene did not affect the RNA and protein expression levels,but the enzyme activity of cells with FUT1 mutations was significantly decreased which resulted in the reduced expressin of H antigen.