猪APOBEC3F基因克隆及真核表达载体的构建与鉴定

Cloning of porcine apolipoprotein B mRNA editing enzyme,catalytic polypeptide-like 3F gene and construction and identification of eukaryotic expression vectors

目的克隆猪载脂蛋白B mRNA编辑酶催化多肽样蛋白3F(apolipoprotein B mRNA editing enzyme,catalyticpolypeptide 3F,APOBEC3F)基因,构建真核表达载体实现其在体外表达与鉴定。方法分离4头21周龄,体质量25～30kg的雌性健康五指山猪的外周血单个核细胞,Trizol法提取细胞总RNA,RT-PCR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鉴定目的基因的表达。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细胞表达,且表达产物定位于细胞质内;Western blot检测示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。

Objective To clone the gene of porcine apolipoprotein B mRNA editing enzyme,catalytic polypeptide-like 3F（APOBEC3F）and construct the eukaryotic expression vectors for its expression and identification in vitro.Methods Peripheral blood mononuclear cells（PBMC） were isolated from blood samples of 4 female healthy Wuzhishan porcine weighing 25 to 30 kg at the age of 21 weeks.RNA was extracted from porcine PBMC using the TRAZO regent.Coding region of the APOBEC3F gene was amplified by RT-PCR.Gene fragments were inserted into the recombinant eukaryotic expression vectors pDsRed1-N1 and modified pCDNA3-Flage-pA3F to construct the eukaryotic expression plasmids of pDsRed1-N1-pA3F and pCDNA3-Flage-pA3F expressed in PK-15 cells.The expression of target gene in PK-15 cells was detected by laser scanning confocal microscopy and Western blotting,respectively.Results The homology of target gene and published gene was 99%.Laser scanning confocal microscopy showed that the target gene was expressed in PK-15 cells and the expressed products were located in the cytoplasm.Western blot analysis revealed that the molecular weight of expressed target protein was consistent with the expected weight.Conclusion The eukaryotic expression vectors of porcine APOBEC3F has been constructed.