Construction of MARC-145ORF6 Cell Line Stably Overexpressing PRRSV M Protein and Its Effect on PRRSV Proliferation
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-145ORF6 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-145ORF6 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-145ORF6 cell line,MARC-145Flag 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-145ORF6 cell li ne 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 type Ⅰ interferon and its related regulatory genes in the cell line;the multi-step growth curve showed that the MARC-145ORF6 cell line promoted the proliferation of PRRSV and increased its viral titer.In conclusion,the MARC-145ORF6 cell line,which can stably express PRRSV M protein,was constructed in this study,and was found to significantly down-regulate the level of type]interferon and promote PRRSV replication.The MARC-145ORF6 cell line constructed in this study will provide an important biological material for the in-depth study of M protein function.
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