人α1,3-岩藻糖基转移酶IV荧光真核表达载体的构建及鉴定  

Construction and identi?cation of human fucosyltransferase 4 eukaryotic fluorenscent expression vector

作　　者：杨雪松[1] 贾爽[1] 张震波[1] 燕秋[1] 

机构地区：[1]大连医科大学生化与分子生物学教研室,辽宁省大连市116044

出　　处：《世界华人消化杂志》2008年第10期1064-1068,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30270329;No.30670465;No.30672753~~

摘　　要：目的:构建并鉴定绿色荧光蛋白为报告基因的pEGFP-N1-FUT4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IV(FUT4)全长基因,克隆至pEGFP-N1-FUT4表达载体并进行鉴定.通过脂质体转染到人表皮癌细胞系A431中,荧光显微镜下观察并经逆转录-聚合酶链反应(RT-PCR)和Western blot检测FUT4的表达.结果:测序及酶切鉴定证明获得人全长FUT4基因,FUT4基因正确插入pEGFP-N1中,在荧光显微镜下观察到绿色荧光蛋白在A431细胞中的表达,RT-PCR和Western blot检测结果显示FUT4的表达明显增加.结论:成功构建绿色荧光蛋白为报告基因的FUT4真核表达载体,并检测到在A431细胞中的表达.AIM： To construct and identify the eukaryotic expression vector pEGFP-N1-FUT4 with enhanced green fluorescent protein （EGFP） gene.METHODS： The full-length fucosyltransferase 4 （FUT4） cDNA was acquired by reverse tran- scription-polymerase chain reaction （RT-PCR） and cloned into pEGFP-Nlvector. The obtained pEGFP-N1-FUT4 was transiently transfected into cell line A431. Then the expression of FUT4 was observed under fluorescence microscope and examined by semi-quantitative RT-PCR and Western blotting. RESULTS： The full-length human FUT4 cDNA was obtained and identified correct through sequencing and enzyme digestion. The recombi- nant plasmid pEGFP-N1-FUT4 was successfully constructed and FUT4 cDNA was correctly in- serted into pEGFP-N1-FUT4. The expression of EGFP in A431 cells transfected with pEGFP-N1- FUT4 was observed by fluorescence microscopy. Semi-quantitative RT-PCR and Western blotting showed that FUT4 expression significantly in- creased after pEGFP-N1-FUT4 transfection in A431 cells in comparison with that in the controis. CONCLUSION： The prokaryotic expression plasmid pEGFP-N1-FUT4 vector is successfully constructed, which could express FUT4 in A431 cells.

关 键 词：α1 3-岩藻糖基转移酶Ⅳ 绿色荧光蛋白 基因克隆 基因表达 逆转录-聚合酶链反应 

分 类 号：R346[医药卫生—基础医学]