稳定表达PRRSV M蛋白的MARC-145^(ORF6)细胞系的构建及其对PRRSV增殖的影响

Construction of MARC-145 ORF6 Cell Line Stably Overexpressing PRRSV M Protein and Its Effect on PRRSV Proliferation

为了给深入研究猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus,PRRSV)ORF6基因编码的M蛋白的生物学功能提供重要试验材料,本研究首先利用慢病毒包装系统构建了过表达PRRSVORF6基因的重组慢病毒质粒,将该质粒连同辅助质粒共同转染至HEK293T细胞获得重组慢病毒;之后将重组慢病毒感染MARC-145细胞,利用嘌呤霉素结合有限稀释法进行筛选,连续筛选3轮后建立了稳定表达PRRSVM蛋白的MARC-145ORF6细胞系;并使用CCK-8试验评估过表达PRRSVM蛋白对MARC-145细胞生长的影响。利用RT-PCR、蛋白免疫印迹(Westernblot)和间接免疫荧光(IFA)评估MARC-145ORF6细胞系的传代稳定性并鉴定M蛋白的亚细胞定位,进一步利用RT-qPCR评估过表达M蛋白对MARC-145细胞的干扰素及相关调节基因的影响;此外,还测定了PRRSV在MARC-145ORF6细胞系、MARC-145Flag细胞系和MARC-145细胞中的病毒滴度并绘制多步生长曲线以比较其差异。CCK-8试验结果表明,过表达PRRSVM蛋白对MARC-145细胞活力无显著影响;RT-qPCR、Westernblot和IFA等试验结果表明,MARC-145ORF6细胞系能够表达PRRSV的M蛋白且在传代过程中稳定。此外,稳定表达PRRSVM蛋白显著下调了细胞系的Ⅰ型干扰素及其相关调节基因;多步生长曲线表明,MARC-145ORF6细胞系促进PRRSV增殖,提高其病毒滴度。综上,本研究构建了可以稳定表达PRRSVM蛋白的MARC-145ORF6细胞系,发现其Ⅰ型干扰素水平显著下调且促进PRRSV复制。本研究构建的MARC-145ORF6细胞系将为M蛋白功能的深入研究提供重要生物材料。

In order to provide important experimental materials for the in-depth study of the biological functions of the M protein encoded by the ORF6 gene of porcine reproductive and respiratory syndrome virus(PRRSV),in this study,we firstly constructed a recombinant lentiviral plasmid overexpressing the ORF6 gene of PRRSV using the lentiviral packaging system.The recombinant lentiviral plasmid overexpressing PRRSV ORF6 gene was constructed using the lentiviral packaging system,and the plasmid,together with the helper plasmid,was co-transfected into HEK293T cells to obtain recombinant lentivirus;after that the recombinant lentiviruses were infected into MARC-145 cells,and screened using puromycin in combination with the finite-dilution method,and after three consecutive rounds of screening,a MARC-145 ORF6 cell line was established that expresses the M protein of PRRSV;and The effect of overexpression of PRRSV M protein on the growth of MARC-145 cells was assessed using CCK-8 assay.RT-PCR,protein immunoblotting(Western blot)and indirect immunofluorescence(IFA)were used to assess the passaging stability of the MARC-145 ORF6 cell line and to identify the subcellular localization of the M protein,and RT-qPCR was further used to assess the effect of overexpression of the M protein on interferon and related regulatory genes in MARC-145 cells;in addition,the effect of PRRSV viral titres in MARC-145 ORF6 cell line,MARC-145 Flag cell line and MARC-145 cells and plotted multi-step growth curves to compare the differences.The results of CCK-8 assay showed that there was no significant effect on the viability of MARC-145 cells after overexpression of PRRSV M protein;the results of RT-qPCR,Western blot and IFA showed that the MARC-145 ORF6 cell line was able to express the M protein of PRRSV and was stable during passaging.And the expression of PRRSV ORF6 gene and M protein were stable.In addition,stable expression of PRRSV M protein significantly down-regulated typeⅠinterferon and its related regulatory genes in the cell line;the multi-step growth curve showed that the MARC-145 ORF6 cell line promoted the proliferation of PRRSV and increased its viral titer.In conclusion,the MARC-145 ORF6 cell line,which can stably express PRRSV M protein,was constructed in this study,and was found to significantly down-regulate the level of typeⅠinterferon and promote PRRSV replication.The MARC-145 ORF6 cell line constructed in this study will provide an important biological material for the in-depth study of M protein function.