猪CD163瞬时转染CHO-K1超表达细胞模型的构建及鉴定  

Construction and identification of porcine CD163 transiently transfected CHO-K1 overexpressing cell model

作　　者：王柳 张泽锴 周焕焕 周傲 张晶 陈洪波 WANG liu;ZHANG Ze-kai;ZHOU Huan-huan;ZHOU Ao;ZHANG Jing;CHEN Hong-bo(School Of Animal Science And Nutritional Engineering,Wuhan Polytechnic University, Wuhan 430023,China)

机构地区：[1]武汉轻工大学动物科学与营养工程学院,湖北武汉430023

出　　处：《武汉轻工大学学报》2021年第2期1-7,共7页Journal of Wuhan Polytechnic University

基　　金：武汉市应用基础前沿项目(2019020701011494);湖北省知识创新专项(自然科学基金一般面上项目,2018CFB535);动物胚胎工程及分子育种湖北省重点实验室开放课题(KLAEMB-2020-03).

摘　　要：猪CD163基因的基因组包括16个内含子、18个外显子,其相关蛋白产物是血红蛋白-触珠蛋白清除剂并可作为病原体受体,在介导感染、维持细胞内环境稳态等方面具有重要功能。本研究旨在构建猪CD163瞬时转染仓鼠卵巢上皮细胞系CHO-K1的超表达细胞模型并对其进行鉴定,以期为CD163分子功能的深入研究奠定基础。研究首先构建pcDNA3.1+-CD163超表达重组质粒,然后使用Lipofectamine 3000转染试剂瞬时转染CHO-K1细胞,6 h后换液并继续连续培养48 h,结合GFP表达情况评估转染效率;收细胞后通过RT-PCR、qRT-PCR和激光共聚焦检测其在mRNA和蛋白水平的CD163表达情况。结果表明,与对照组相比转染后CHO-K1细胞中猪CD163在mRNA水平和蛋白水平的表达量方面均显著上调;亚细胞定位结果显示,超表达的猪CD163蛋白主要定位在CHO-K1细胞膜,同时胞质中也有一定分布。研究成功构建了超表达猪CD163的CHO-K1瞬时转染细胞模型,为下一步探究猪CD163介导细菌或病毒性病原体感染的生物学功能奠定了基础。Porcine CD163 gene comprises 16 introns and 18 exons.The CD163 protein acts as a hemoglobin-haptoglobin scavenger and pathogen receptor,playing important roles in mediating infections and maintaining intracellular environment homeostasis.The aim of this study was to construct and characterize an overexpression cell model of porcine CD163 that transiently transfected hamster ovarian epithelial cell line CHO-K1,and to pave a way for the following investigation of CD163 molecular functions.An overexpression recombinant plasmid,named pcDNA3.1+-CD163 was firstly constructed,then it was transiently transfected into CHO-K1 by using Lipofectamine 3000 transfection reagent based on transfection efficiency evaluation via GFP expression.Cells were continuously cultured for 48 h after the first 6 h culture with the transfection reagent.In comparison of control,both RT-PCR and qRT-PCR experiments showed that the expression of porcine CD163 was significantly up-regulated at mRNA level in CHO-K1 CD163+cells;and its overexpression at protein level was further validated with Laser Scanning Confocol(LSC)detection.The subcellular localization analysis revealed that the overexpressed porcine CD163 protein was mainly localized in the CHO-K1 cell membrane,and also had some distribution in the cytoplasm.In conclusion,this study successfully constructed a transient transfection cell model of CHO-K1 overexpressing porcine CD163,which laid the foundation for further investigation of its functions in bacterial or viral infections.

关 键 词：猪 CD163 CHO-K1 转染 表达 

分 类 号：S821.5[农业科学—畜牧学]