稳定表达PRRSV GP2蛋白的Marc-145细胞系的构建

Construction of a Marc-145 Cell Line Stably Expressing PRRSV GP2 Protein

尽管在全球范围内努力控制猪繁殖与呼吸综合征病毒(PRRSV)的感染,但该病毒持续在全世界的养猪业中引起经济问题。本研究以PRRSV全长感染性克隆pJXwn06为模板,利用PCR方法扩增ORF2基因序列,并克隆至慢病毒载体中,获得重组质粒pLV-EF1a-EGFP-2A-GP2。通过慢病毒包装系统获得重组的慢病毒颗粒,并将其转导至Marc-145细胞,用含有嘌呤霉素的培养基进行筛选,初筛后的细胞采用有限稀释法进行克隆,最终获得细胞系,并通过PCR、Western blot试验验证了细胞系中GP2蛋白的稳定表达。本实验成功建立了稳定表达PRRSV GP2蛋白的Marc-145细胞系,为研究GP2蛋白免疫学特性和结构功能奠定了基础。

Despite global efforts to control the infection of Porcine reproductive and respiratory syndrome virus(PRRSV),the virus continues to cause economic problems in the swine industry worldwide.In this study,the PRRSV full-length infectious clone pJXwn06 was used as a template,and the ORF2 gene sequence was amplified by PCR and cloned into a lentiviral vector to obtain the recombinant plasmid pLV-EF1a-EGFP-2A-GP2.Recombinant lentiviral particles were obtained through the Lentivirus packaging system and were transduced to Marc-145 cells.Then,the cells with the particles were screened with puromycin-containing medium and cloned by the limiting dilution method to finally obtain the cell line.As a result,the stable expression of GP2 protein in the cell line was confirmed by PCR and Western blot.In this experiment,a Marc-145 cell line stably expressing PRRSV GP2 protein was successfully established,which laid a foundation for the study of the immunological characteristics,structure and function of the GP2 protein.