期刊论文

Establishment and application of immortalized porcine retinal pigment epithelial cells

中文

西北农林科技大学学报(自然科学版)

1671-9387

2024

52

2

22-31

10.13207/j.cnki.jnwafu.2024.02.003

2021JJ30060:湖南省自然科学基金面上项目 cxcy2022001:国家级大学生创新创业训练计划项目 :海南省农业科学院重点实验室资助项目 WST2018014:山东省外专双百计划项目

本校为第一机构

[目的]建立永生化猪视网膜色素上皮细胞系,为病毒学研究提供一种新的细胞材料.[方法]合成猪腺病毒3型E1基因,将其克隆至真核表达质粒pCI-neo中,构建E1基因真核表达质粒pCI-neo-E1,进行PCR和测序鉴定.提取pCI-neo-E1质粒,转染原代猪视网膜色素上皮细胞,用新霉素G418筛选猪永生化视网膜色素上皮细胞系(RPECs).检测RPECs的角蛋白18和19,通过细胞计数测定其生长曲线,流式细胞仪检测细胞周期,并对其进行染色体核型分析.分别将猪流行性腹泻病毒(PEDV)CV77毒株、猪圆环病毒2型(PCV2)DBN-SX07株和猪伪狂犬病毒(PRV)Bartha-K61株接种RPECs,同时将PEDV CV77毒株接种绿猴肾细胞(Vero细胞),将PCV2 DBN-SX07株...

[Objective]This study established an immortalized porcine retinal pigment epithelial cell line to provide a new cell material for virological research.[Method]The E1 gene of porcine adenovirus type 3 was synthesized and cloned into the eukaryotic expression plasmid pCI-neo.The eukaryotic expres-sion plasmid pCI-neo-E1 was constructed and identified by PCR and sequencing.The pCI-neo-E1 plasmid was extracted and transfected into primary porcine retinal pigment epithelial cells.Porcine immortalized retinal pigment epithelial cell lines(RPECs)were selected by neomycin G418.Keratins 18 and 19 were ...