猪apobec3f基因扩增与真核表达载体的构建及鉴定.docVIP

猪APOBEC3F基因克隆及真核表达载体的构建与鉴定 孙 宇1,2，罗 佳3，马玉媛1，章金刚1 （100850北京 军事医学科学院野战输血研究1；解放军第305医院检验科2；广西大学动物科学技术学院3） [摘要]目的 克隆猪载脂蛋白B mRNA编辑酶催化多肽样3F（apolipoprotein B mRNA editing enzyme，catalytic polypeptide 3F，APOBEC3F）基因4头的外周血单个核细胞，TRIZOL法提取细胞总RNA，RT-PCR技术特异性扩增目的基因，目的基因插入真核表达载体pDSred1-N1及改造的pCDNA3-Flage载体构建表达质粒并在PK-15细胞中进行表达。激光共聚焦显微镜及Western blot方法对目的基因的表达进行鉴定。结果 克隆的目的基因与公布的基因序列同源性达99%；激光共聚焦显微镜显示目的基因在PK-15细胞中实现了表达，且表达产物定位于细胞质内；Western blot鉴定结果表明，表达的目的蛋白大小与预期相符。结论 成功构建了猪APOBEC3F真核表达载体，为进一步研究其与猪内源性反转录病毒（Porcine endogenous?retrovirus，PERV）相互作用奠定了基础。 [关键词]APOBEC3F；克隆；真核表达载体；鉴定 The cloning of Porcine APOBEC3F gene and the construction and identification of eukaryotic expression vectors Sun Yu1，2，Ma Yuyuan1，Luo Jia3，Zhang Jingang1（1Institute of Transfusion Medicine， Academy of Military Medical Sciences， Beijing 100085；2The 305 Hospital of PLA Laboratory Department，Beijing 100017；3College of Animals Sciences Technology, Guangxi University, Nanning 530005.） [Abstract] Objective To clone the gene of porcine apolipoprotein B mRNA editing enzyme，catalytic polypeptide-like 3F （APOBEC3F） and construct the eukaryotic expression vector for the expression and identification in vitro. Methods Separating the Peripheral blood mononuclear cells (PBMCs) from the bloods of four female healthy Wuzhishan porcine which ages were 21 weeks and weight were 25~30kg , then extracting the RNA from the porcine PBMC by the TRAZO regent. The coding region of APOBEC3F gene was amplified by RT-PCR， and then the gene fragment was inserted into the recombinant eukaryotic expression vector pDSred1-N1 and pCDNA3-Flage-pA3F which had been transformed to construct the eukaryotic expression plasmids of pDSred1-N1-pA3F and pCDNA3-Flage-pA3F， which were trans into PK-15 cell by Lip2000 transfection agent， respectively. The expressed products in the pk-15 cells were detected by Fluorescence detection by the Laser scanning confocal microscope and Western blot. Results The Homologous of the gene which had b