Identification And Function Of Porcine Circovirus Type 2 Encoding Protein 7

Porcine circovirus type 2(PCV2)is the causative agent of Porcine circovirus diseases(PCVDs),mainly infecting the immune cells of infected pigs,resulting in severe immunosuppression.This increases the susceptibility of infected pigs to other pathogens,causing a variety of diseases,seriously endangering the health of pig breeding and the prevention and control of pig diseases.PCV2 is the smallest DNA virus that has been found to infect mammals.The genome consists of 1766-1768 nucleotides.The elucidation of the pathogenic mechanism of the virus is the basis of disease prevention and control.The role of the encoded protein in the pathogenicity of the virus is revealed an important aspect of its pathogenic mechanism.The genome structure of PCV2 is simple.Bioinformatics software analysis found that there are 11 potential open reading frames(ORFs)in the genome,which encode the structural and non-structural proteins of the virus,and play a role in the pathogenesis of the virus.Only 6 ORFs(ORF1-6)were found.The discovery and identification of new ORFs will add new members to the etiology of PCV2,clarify its role in viral pathogenicity,and provide new scientific information for the discovery of the pathogenic mechanism of PCV2.We found that there is a conserved ORF in the PCV2 genome,contained in the ORF2 gene,at 1524-1033 nucleotides(492 nt)in the genome,theoretically encoding 164 amino acids,and predicting that the molecular weight of the protein is about 18 kDa.In order to connect with the naming of PCV2 ORFs that have been reported,it is temporarily named "ORF7".In this study,the expression of ORF7 in PCV2 infected cells was tested.On the basis of confirming the existence of ORF7,the effect of ORF7 on the host cell cycle,apoptosis,expression of cellular innate immune factors and the replication of PCV2 was carried out.The following results were obtained:(1)Preparation of murine anti-ORF7 protein polyclonal antibody.Constructed pGEX-6p-1-ORF7 recombinant plasmid and induced expression by prokaryotic expression system.It was found that the highest protein expression was induced by IPTG 0.8 mmol/L and 37? for 12 h,and it was mainly expressed in bacterial pellet,The size of pGEX-6p-1-ORF7 recombinant protein is about 44 kDa.The pGEX-6p-1-ORF7 recombinant protein was purified by excising and mixed with adjuvant.After immunizing Balb/c mice 4 times,the positive serum titer of the mice detected by ELISA was 1:10 000;it can specifically detect the expression of ORF7 protein after PCV2 infection in PK-15 cells by IFA test;Western blot test can specifically recognize the expression of pGEX-6p-1-ORF7 and pCDH-CMV-Flag-ORF7 recombinant protein.It indicates that the prepared polyclonal antibody can be used for the detection of ORF7 protein.(2)Detection and identification of ORF7 in PCV2 infected PK-15 cells.Through RT-PCR and RT-qPCR detection,it was found that PCV2 infected PK-15 cells at different time points,and ORF7 mRNA could be detected at different time points;Western blot detection found that ORF7 protein expression was present in PCV2 infected cells.The results confirmed the existence of ORF7 gene transcription and protein expression in the process of PCV2 sensory cells.(3)The effect of ORF7 protein on the function of PK-15 cells.PK-15 cells were transfected with pCDH-CMV-Flag-ORF7 recombinant plasmid.RT-qPCR detection showed that the transcription of endoplasmic reticulum stress marker protein GRP78 gene was significantly up-regulated 36 h and 48 h after transfection;It was found that the activity of the cells increased from 12 h to 48 h after transfection by CCK-8 method.Flow cytometry showed that the number of transfected cells in the S phase was significantly reduced,and the percentage of apoptotic cells was reduced.RT-qPCR and Western blot detection found that the expression of Bcl-2 and Bax were up-regulated,the Bcl-2/Bax value was increased,and the expression of Caspase-8/3 was down-regulated,suggesting the ORF7 protein has the effect of inhibiting apoptosis;RT-qPCR,Western blot and ELISA detection found that the expression of TNF-?,NF-?B,IL-10 and IFN-? in transfected cells was up-regulated,and the expression of IL-2 and IL-12 was down-regulated.After PCV2 infection of transfected cells,fluorescence microplate reader found that the ROS level of transfected cells was reduced compared with PCV2 infected control cells.The above research results indicate that ORF7 protein inhibits host cell apoptosis,promotes the secretion of innate immune factors,and reduces cellular ROS caused by PCV2.(4)Detection of virus replication in PK-15 cells expressing ORF7 protein after infection with PCV2.The pCDH-CMV-Flag-ORF7 plasmid was transfected into PK-15 cells and then infected with PCV2.RT-qPCR detection showed that the PCV2 Cap gene mRNA was reduced in the transfected cells compared with control cells from 12 h to 48 h after infection.The results suggest that cells expressing ORF7 protein after inoculation with PCV2 will down-regulate Cap gene transcription.This study identified the expression of ORF7 in PCV2-infected cells from the level of gene transcription and protein translation;the vitro studies found that ORF7 protein can promote endoplasmic reticulum stress response in PK-15 cells,shorten the cell cycle,inhibit cell apoptosis,affect innate immune response,impact on PCV2 replication.The research results provide a new perspective and scientific data for the elucidation of the pathogenic mechanism of PCV2.