小鼠α1,2岩藻糖基转移酶基因的克隆及表达

CLONING AND EXPRESSION OF MURINE α1,2-FUCOSYLTRANSFERASE GENE

本文报告小鼠GDP岩藻糖:β半乳糖苷α1,2-岩藻糖基转移酶(α1,2-fucosyltansferase,α1,2- FT)基因的克隆,并进行功能鉴定。利用RT-PCR方法克隆小鼠α1,2-岩藻糖基转移酶基因编码区 MFUT-Ⅱ,测序后将其插入表达载体pcDNA3．1的多克隆位点,构建表达载体pcDNA3．1-MFUT-Ⅱ; 采用磷酸钙法将其转染于COS-7细胞进行表达,通过对底物特异性比较研究酶的性质;应用 Northern印迹杂交法研究基因在小鼠组织中的表达情况;应用southern印迹杂交法分析基因存在状态。结果证实MFUT-Ⅱ为小鼠α1,2-岩藻糖基转移酶基因家族的新成员,含有一个完整的开放读码框,可编码347个氨基酸．其估计分子质量为39 kDa,和小鼠H及Secl基因具有序列同源性,分别与人类Se基因(79．0％)、大鼠Rat FTB(89％)基因、兔Rabbit FT-Ⅲ基因(77％)具有较高的序列同源性。用MFUT-Ⅱ基因转染的COS-7细胞具有α1,2-FT活性,MFUT-Ⅱ可在多种组织中产生 3．5kb大小的mRNA转录产物。基因Southern印迹杂交分析结果显示:基因MFUT-Ⅱ仅为一个拷贝。这些结果证明MFUT-Ⅱ为小鼠的Se基因。

MFUT-II, a DNA fragment encoding murine α1,2-fucosyhransferase,was cloned by RT-PCR. The open-reading frame was ligated into mammalian expression vector pcDNA 3.1 （pcDNA3.1-MFUT-11 ） and was transiently transfected into COS-7 cells using a Cellphect Transfection kit. Moreover, the expression of the gene in murine tissues was analyzed with Northern hybridization showing a 3.5-Kb mRNA transcript product in several tissues. The existence of MFUT-Ⅱ was detected by Southern blot. MFUT-Ⅱ was found to be a new member of the murine α1,2-FT gene family. Its open reading frame encoded 347 amino acids with a predicted molecular mass of 39.21 kDa and exhibited sequence homology with murine H ＆ Secl gene. MFUT-Ⅱ also exhibited sequence homology with Human Se gene （79.0%）, Rat FTB gene （89.0%） and Rabbit FT-Ⅲ gene （77.0%）, respectively. COS-7 cells transfected with pcDNA3.1-MFUT-11 showed alpha 1,2-fucosyhransferase activity. Southern analysis revealed that MFUT-Ⅱ was present in the mouse genome as a single-copy gene. In conclusion, MFUT-Ⅱ was a murine Se gene.