猪CD163 cDNA扩增及其稳定表达细胞系(CHO-K1)的建立  

Amplification of porcine CD163 cDNA and establishment of CHO-K1 cell line stably expressing CD163

作　　者：徐建[1,2] 霍珊珊[3] 张建楼[3] 张永红[3] 崔丹[3] 仲飞[2,3] 李秀锦[1] 

机构地区：[1]燕山大学环境与化学工程学院生物工程系,河北秦皇岛066004 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [3]河北农业大学动物医学院基础兽医系/河北兽医生物技术工程技术研究中心,河北保定071001

出　　处：《河北农业大学学报》2016年第4期88-92,122,共6页Journal of Hebei Agricultural University

基　　金：河北省自然科学基金(C2013204130);家畜疫病病原生物学国家重点实验室开放课题(SKLVEB2013KFKT022);河北省高等学校科学技术研究项目(ZD20131056)

摘　　要：猪CD163是猪繁殖与呼吸综合征病毒(PRRSV)的重要受体之一。为探讨此受体在病毒感染过程中与PRRSV的相互作用,需要建立稳定表达猪全长CD163基因的细胞系。首先通过RT-PCR方法从猪肺泡巨噬细胞中扩增猪全长CD163cDNA,然后通过KpnI和XhoI将其插入到pcDNA3.1A质粒中,构建成真核表达载体。将表达载体转染CHO-K1细胞,通过G418加压,筛选出在细胞膜上稳定表达猪CD163的细胞系。结果显示,扩增出的猪CD163基因与基因库的序列基本一致,个别碱基的突变没有引起编码氨基酸的改变。通过转染表达载体和G418筛选,成功构建了稳定表达猪全长CD163基因的CHO-K1细胞系。经流式细胞仪检测证实,重组猪CD163可在CHO-K1细胞膜上进行表达,这为进一步研究PRRSV与CD163受体相互作用提供了必要条件。CD163is one of the important receptors of porcine reproductive and respiratory syndrome virus（PRRSV）.To investigate the interaction between PRRSV and CD163 during PRRSV infection,the cell line for stably expressing CD163 was required.Firstly,the fulllength porcine CD163 cDNA was amplified from porcine alveolar macrophages by RT-PCR andwas inserted into the eukaryotic expression vector pcDNA3.1A by KpnI and Xho I to construct expression vector of CD163 gene.The CD163 vector was transfected into CHO-K1 cells and the CD163-stably-expressed cells were selected by G418.The results showed that the amplified CD163 gene was basically consistent with that in Genbank although there were some base substitutions which,however,did not result in amino acid changes.The CD163-stably-expressed CHO-K1 cell line was successfully established by transfection of the vector and selection with G418.Flow cytometry assay indicated that the recombinant porcine CD163 could be expressed on the CHO-K1 cell membrane,which provided necessary conditions for further investigating the interaction between PRRSV and CD163 receptor.

关 键 词：猪CD163 稳定表达细胞系 CHO-K1细胞 流式细胞术 

分 类 号：S828[农业科学—畜牧学]