畜牧与兽医2024，Vol.56Issue(12)：74-80,7.
PRRSV非结构蛋白NSP4及其3个主要结构域过表达慢病毒载体的构建及鉴定
Construction and identification of lentiviral vectors overexpressing PRRSV NSP4 and its three major domains
摘要
Abstract
The aims of this study were to construct lentiviral vectors for porcine reproductive and respiratory syndrome virus(PRRSV)NSP4 and its three domains,and to package the lentiviruses and detect the expression of the four lentiviruses in THP-1 and PAM.The plas-mid pXJ41-HA-NSP4 was used as a template to amplify the NSP4,NSP4-DI,NSP4-DII,and NSP4-DIII genes.The amplified gene frag-ments were ligated into the pCD513B lentiviral vector,and the recombinant plasmids obtained were named pCD513B-NSP4,pCD513B-NSP4-DⅠ,pCD513B-NSP4-DⅡ and pCD513B-NSP4-DⅢ,respectively.HEK-293T cells were then transfected with the lentiviral re-combinant plasmids together with packaging plasmids pLP1,pLP2,pLP/VSVG,and the lentiviruses obtained by packaging were named rLV-NSP4,rLV-NSP4-DⅠ,rLV-NSP4-DⅡ and rLV-NSP4-DⅢ,respectively.The four recombinant lentiviruses were infected into HEK-293T cells,and the lentiviral titers were determined.Finally,THP-1 cells and PAM cells were infected respectively,the expression of target proteins was observed by fluorescence microscopy,the transcription level of target genes was detected by qPCR,and the expression level of target proteins was detected by Western blot at different infection times.The results showed that the viral titers of the packaged rLV-NSP4,rLV-NSP4-DⅠ,rLV-NSP4-DⅡ,and rLV-NSP4-DⅢ were 2.2×106 TU/mL,2.8×106 TU/mL,2.5×106 TU/mL and 2.5×106 TU/mL.The results of fluorescence microscopy showed that the number of positive cells increased with the increase in time after the four lentiviruses infected the THP-1 cells and PAM cells.qPCR results showed that the transcript levels of the target genes increased with the in-crease in infection time within 60 h after lentivirus infection.The Western blot results showed that the four lentiviruses were able to success-fully infect THP-1 and PAM cells and express the corresponding target proteins,and that the protein expression levels increased with the in-crease in infection time.In conclusion,the recombinantly overexpressed lentivirus of PRRSV NSP4 and its three main domains were success-fully packaged and expressed here,which laid the foundation for further research on the function of PRRSV NSP4 and its three domains.关键词
猪繁殖与呼吸综合征病毒/NSP4/慢病毒载体/鉴定Key words
PRRSV/NSP4/lentiviral vector/identification分类
农业科技引用本文复制引用
李子镛,任金瑞,吴香菊,丛晓燕,李均同,齐静,王晓晔,胡悦,杜以军..PRRSV非结构蛋白NSP4及其3个主要结构域过表达慢病毒载体的构建及鉴定[J].畜牧与兽医,2024,56(12):74-80,7.基金项目
山东省自然科学基金项目(ZR2023MC076,ZR2021ZD08,ZR2021MC139) （ZR2023MC076,ZR2021ZD08,ZR2021MC139）
国家自然科学基金项目(32373094,32102710) （32373094,32102710）
国家兽用生物制品工程技术研究中心开放课题[GTKF(23)009] （23）
