鸡marco稳转巨噬细胞系的构建与鉴定

Construction and Identification of the Chicken marco-stable Macrophage Cell Line

旨在通过慢病毒表达系统构建稳定表达marco基因的HD11细胞系,为研究marco基因的抗病毒免疫功能提供重要工具。首先,以HD11细胞为模板,通过PCR扩增获得marco基因编码区序列,并将其克隆到慢病毒载体上,获得重组慢病毒质粒pLV3-CMV-MCS-Puro-marco;其次,利用四质粒慢病毒包装系统将重组质粒pLV3-CMV-MCS-Puro-marco与辅助质粒pLP1、pLP2、pMD2.G共转染至293T细胞进行慢病毒包装,构建含鸡marco基因的重组慢病毒。最后,用包装成功的重组慢病毒感染HD11细胞72 h后进行嘌呤霉素(2μg·mL^(-1))筛选,通过RT-qPCR检测marco基因表达后最终确认获得稳定表达鸡marco基因的HD11细胞系。本研究成功构建了稳定表达鸡marco基因的HD11细胞系,为后续深入研究marco基因的功能提供了重要基础。

The study aimed to establish the HD11 cell line with a stable expression of the marco gene through the lentivirus expression system,providing a crucial tool for investigating the antiviral immune function of the marco gene.Initially,the marco gene coding sequence was amplified by PCR using HD11 cells as a template and cloned into the lentiviral vector to generate the recombinant lentiviral plasmid pLV3-CMV-MCS-Puro-marco.Subsequently,the recombinant plasmid pLV3-CMV-MCS-Puro-marco was co-transfected with helper plasmids pLP1,pLP2,and pMD2.G into 293T cells using a tetra-plasmid lentivirus packaging system,resulting in the construction of recombinant lentivirus carrying the chicken marco gene.The successfully packaged recombinant lentivirus was then used to infect HD11 cells,followed by puromycin(2μg·mL^(-1))selection for 72 h.The stable expression of the chicken marco gene in the HD11 cell line was confirmed by RT-qPCR analysis,indicating the successful establishment of a stable cell line expressing the chicken marco gene.This study successfully established a stable HD11 cell line expressing the chicken marco gene,providing a crucial basis for further exploration of the marco gene’s function.