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

旨在构建猪繁殖与呼吸综合征病毒(PRRSV)NSP4蛋白及其3个主要结构域的过表达慢病毒载体,包装慢病毒并检测4种慢病毒在人单核细胞白血病细胞THP-1和猪肺泡巨噬细胞PAM中的表达。试验以质粒pXJ41-HA-NSP4为模板,分别扩增NSP4全长及其3个结构域NSP4-DⅠ、NSP4-DⅡ、NSP4-DⅢ片段,连接到pCD513B慢病毒表达载体中,构建重组质粒pCD513B-NSP4、pCD513B-NSP4-DⅠ、pCD513B-NSP4-DⅡ、pCD513B-NSP4-DⅢ;将慢病毒重组质粒与包装质粒pLP1、pLP2、pLP/VSVG共转染HEK-293T细胞,包装获得4种重组慢病毒rLV-NSP4、rLV-NSP4-DⅠ、rLV-NSP4-DⅡ、rLV-NSP4-DⅢ,并感染HEK-293T细胞测定病毒滴度;分别将4种重组慢病毒感染THP-1细胞、PAM细胞,荧光显微镜观察目的蛋白表达,荧光定量PCR(qPCR)检测目的基因转录水平,Western blot检测不同感染时间目的蛋白表达水平。结果:4种慢病毒rLV-NSP4、rLV-NSP4-DⅠ、rLV-NSP4-DⅡ、rLV-NSP4-DⅢ感染HEK-293T细胞的病毒滴度分别为2.2×10^(6)、2.8×10^(6)、2.5×10^(6)和2.5×10^(6) TU/mL;荧光显微镜观察显示4种慢病毒感染THP-1细胞和PAM细胞后阳性细胞数均随时间的增加而增多;qPCR结果表明目的基因转录水平在慢病毒感染后60 h内随着感染时间增加而升高;Western blot表明4种慢病毒能够成功感染THP-1细胞和PAM细胞并稳定表达相应的目的蛋白,且蛋白表达水平随着感染时间增加而升高。综上,本研究成功包装PRRSV NSP4及其3个主要结构域重组过表达慢病毒,为进一步研究PRRSV NSP4及其3个结构域的功能奠定了基础。

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,pCD513BNSP4-DⅠ,pCD513B-NSP4-DⅡand 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-DⅡand 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 rLVNSP4,rLV-NSP4-DⅠ,rLV-NSP4-DⅡ,and rLV-NSP4-DⅢwere 2.2×10^(6) TU/mL,2.8×10^(6) TU/mL,2.5×10^(6) TU/mL and 2.5×10^(6) 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.