三黄鸡α-2,3唾液酸转移酶Ⅲ基因的克隆及其在293T细胞中的表达  

Gene cloning of α-2,3 sialyltransferase III of Chinese yellow chicken and its expression in 293T cells

作　　者：崔连成[1] 周飞[1] 黄志强[1] 童双[1] 黄冬妮[1] 袁远华[1] 宁章勇[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《畜牧与兽医》2013年第4期11-13,共3页Animal Husbandry & Veterinary Medicine

基　　金：广东省自然科学基金(7300744)

摘　　要：唾液酸转移酶催化机体唾液酸的形成,与流感病毒感染的种属特异性具有密切的关系。以三黄鸡法氏囊组织总RNA为模板,运用RT-PCR方法扩增了三黄鸡α-2,3唾液酸转移酶Ⅲ(ST3GalⅢ)基因。将其克隆到带有加强型绿色荧光蛋白(EGFP)报告基因的pIRES2真核表达载体中,对重组质粒pIRES2-EGFP-ST3GalⅢ进行酶切和测序鉴定后,采用阳离子脂质体转染法将重组质粒转染到293T细胞,荧光显微镜和West-ern blot检测观察其表达情况。结果表明,转染pIRES2-EGFP-ST3GalⅢ质粒后的细胞,在荧光显微镜下检测到绿色荧光信号,并且通过Westernblot进一步验证了ST3GalⅢ在细胞中的表达。Sialyhransferases are the enzymes which catalyze the biosynthesis of the sialic acid, and this process is closely related to host specificity of influenza virus. The gene of α-2, 3 sialyltransferase III（ ST3Gal III）, of Chinese Yellow Chicken was amplified by RT-PCR method with the total RNA extracted from bursa of fabricius tissue. To construct the expression vector pIRES2-EGFP-ST3Gal III, the ampli- fied gene was cloned into the vector pIRES2-EGFP with the reported gene EGFP （enhanced green fluorescence protein）. The positive recom- binant pIRES2-EGFP-ST3Gal III was transfected into 293 T cells by liposomes. The expression of the recombinant pIRES2-EGFP-ST3Gal III in the 293 T cells was observed by fluorescence microscope and tested by Western blot. The result indicated that ST3GalIII was successfully expressed in the 293 T cells.

关 键 词：唾液酸转移酶 三黄鸡 真核表达 

分 类 号：Q51[生物学—生物化学]