PRRSV Nsp4基因突变株对β2M表达的影响OA北大核心CSTPCD

[Objectives]This study was conducted to investigate whether porcine reproductive and respiratory syndrome virus(PRRSV)Nsp4 gene played a key role in the inhibition of β2M expression,which laid a foundation for further study of the immunosuppressive mechanism of PRRSV on antigen presentation.[Methods]In this experiment,the reverse genetic platform of PRRSV was established,and the wild PRRSV strain SH2020 was successfully saved.Based on the previous research on different domains of Nsp4 gene and the prediction of amino acid site function,three and five different amino acid sites in the Domain Ⅱ and Domain Ⅲ of NSP4 gene were screened,and different amino acid substitution mutations were performed on viral infectious cloning plasmids.The constructed infectious clone plasmids were then transfected into Marc-145 cells to rescue Nsp4 mutant PRRSV viruses.The rescued PRRSV virus was used to infect PAM cells at a MOI of 0.02,and RT-qPCR and Western blot were used to verify whether PRRSV Nsp4 gene could play a role in the expression of β2M in PAM cells.[Results]The mutant strain D2-5(Nsp4-A80L)was rescued successfully,the character of growth kinetics of which was similar to that of wild strain SH2020;but other mutant sites of Nsp4 gene led the restriction to the replication of the PRRSV which was not accessible to the experiment assay.It was verified that D2-5 strain could enhance the expression of the β2M on the Marc-145 and PAM through the Western blot and RT-qPCR while the wild strain SH2020 presented the inhibition to the β2M.[Conclusions]Nsp4 gene plays an important role in inhibiting the expression of β2M molecule in PRRSV cells,and Nsp4-A80L mutation can significantly promote the expression of β2M.