人α1,3-岩藻糖基转移酶VII荧光真核表达载体的构建及鉴定

目的:构建并鉴定绿色荧光蛋白为报告基因的pIRES2-EGFP-FUT7真核表达载体。方法:采用RT-PCR方法获得α1,3-岩藻糖基转移酶VII(FUT7)全长基因,克隆至pMD18-TSimple载体后,将FUT7亚克隆至pIRES2-EGFP表达载体并进行鉴定。通过脂质体转染到子宫内膜RL95-2细胞中,荧光显微镜下观察并经RT-PCR检测FUT7的表达。结果:测序及酶切鉴定证明获得人全长FUT7基因,FUT7基因正确插入pIRES2-EGFP中,在荧光显微镜下观察到了绿色荧光蛋白在RL95-2细胞中的表达,半定量RT-PCR检测结果显示FUT7的表达明显增加。结论:成功构建了绿色荧光蛋白为报告基因的FUT7真核表达载体,并检测到在RL95-2细胞中的表达,为研究FUT7及其合成的糖抗原sLex在胚胎着床中的作用奠定了基础。

Construction and identification of human FUT7 eukaryotic fluorenscent expression vector

Objective: To construct and identify the eukaryotic expression vector pIRES2-EGFP-FUT7 with EGFP gene. Methods: The full-length FUT7 cDNA was acquired by RT-PCR and cloned into pMD18-T simple vector. Then the product was subcloned into pIRES2-EGFP and the vector was identified by sequencing and cleavage with restriction endonucleases. The pIRES2-EGFP-FUT7 was transiently transfected into cell line RL95- 2. Then the expression of FUT7 was observed under fluorescence microscope and examined by semi-quantitatve RT-PCR. Results: The full-length human FUT7 cDNA was obtained. The recombinant plasmid pIRES2-EGFP- FUT7 was successfully constructed and FUT7 cDNA was correctly inserted into pIRES2-EGFP. The expression of EGFP in RL95-2 cells transfected with pIRES2-EGFP-FUT7 was observed by fluorescence microscope. The semiquantitative RT-PCR showed that the expression of FUT7 significantly increased after PIRES2-EGFP-FUT7 transfection in RL95-2 compared with that of the control. Conclusion: It is important to construct the eukaryotic expression vector pIRES2-EGFP-FUT7 harbouring full-length human FUT7 cDNA with EGFP and transfect them into RL95-2 cells. The work provides the basis for the investigation of the roles of FUT7 gene and sLex in embryo implantation