中文摘要：

试验旨在构建一株高效表达猪CD163（pCD163）的Marc-145细胞系,为猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus,PRRSV）的临床分离和疫苗生产奠定基础。根据GenBank中序列设计引物从猪肺泡巨噬细胞（PAM）中扩增pCD163基因,将其插入真核表达载体pCI-neo构建真核表达质粒pCIpCD163,将该重组质粒转染Marc-145细胞,通过G418筛选、单克隆化并扩大培养筛选获得表达pCD163的Marc-145细胞系,IFA、Western blotting鉴定其表达情况。IFA结果显示,构建的pCD163-Marc细胞系中荧光明显亮于普通Marc-145细胞;Western blotting结果显示,pCD163-Marc细胞系中CD163蛋白表达量约为对照Marc-145细胞中CD163蛋白表达量的8.7倍。且该细胞系可稳定传至20代,各代次之间表达量无差异。证明高效表达猪CD163的Marc-145细胞系构建成功。

英文摘要：

This study was attempted to generate one Marc-145 cell line stably and highly express- ing porcine CD163 （pCD163） and set the foundation for PRRSV isolation and vaccine production. CD163 was shown to be a cellular receptor capable of mediating infection of PRRSV non-permis- sive cell lines. The pCD163 gene was amplified by RT-PCR from porcine alveolar macrophages and cloned into the eukaryotic expression vector pCI-neo,then the positive plasmid pCI-pCD163 was transfected into Marc-145 cells. After selecting with G418 and subcloning for 3 times,Marc- 145 cell line expressing pCD163 was established. IFA results indicated that the fluorescence of pCD163-Marc cells was significantly brighter than Marc-145 cells ~ Western blotting results indica- ted that the pCD163-Marc cells could express higher levels of CD163 and the expression level was 8.7 times higher than Marc-145 cells. The pCD163-Mare cell line could be stably passaged for 20 passages and the expression level of CD163 was similar with different passages,which would be a valuable tool for facilitating virus propagation and vaccine production.