Preliminary Study On Gene Function ORF6 Of Porcine Circovirus Type2

Porcine circovirus type 2(PCV2) infection are recognized as the major factors in a variety of diseases, such as postweaning multisystemic wasting syndrome(PMWS), porcine dermatitis and nephropathy syndrome(PDNS), porcine proliferative and necrotizing pneumonia.PCV2 is widely spreaded in the world, leads to enormous economic losses in swine production, while the pathogenic mechanism is not clear. It is predicted that PCV2 contains 11 open reading frames(ORF). Currently, five proteins of the virus had been identified. In this study, we identified a new protein, encoded by ORF6 gene. ORF6 is 90 bp in length and located in 1611-1522 bp of negative-strand. It encodes 30 aa with 2.8KD.In this study, we identified the ORF6 protein of PCV2. Then the function and biology ORF6 were studied.The results is as following:1. Prokaryotic expression of PCV2 ORF6 and preparation of polyclonal antibody The prokaryotic expression plasmid p GEX-2ORF6 was generated by ligating two copies of the complete ORF6 genome in tandem into the p GEX-KG vector. The plasmid was transformed into E.coli, and induced by IPTG to get GST-2ORF6 fusion protein in a soluble form. Then the protein was purified, andimmuned rabbits to prepare polyclonal antibody.2. Identification of transcription and expression of PCV2 ORF6 ORF6 gene is completely contained in ORF2, in order to verify the transcription of ORF6 m RNA, we detected the difference of m RNA between ORF2+6 and ORF2 in PK-15 cells infected by PCV2 through fluorescence quantitative PCRPCV2 was inoculated into PK-15 cells. Total RNAs were extracted at different infected time and ORF2+6 and ORF2 m RNA were analyzed. The copies of ORF2+6 were significantly higher than the ORF2 transcript copies at different times postinfection. The fold differences of the ORF2+6 versus ORF2 m RNA were varied 2.1-3 over time.The expression of ORF6 protein was identified in PCV2-infected PK-15 cells by indirect immunofluorescence(IFA) using ORF6 polyclonal antibody. The results showed that the specific green fluorescence was observed in PCV2 infected cells, which indicated that ORF6 protein was expressed. We identified the ORF6 protein of PCV2 by detecting the transcription and protein expression of ORF6 gene.3. Subcellular location and eukaryotic expression of PCV2 ORF6PCV2 WUH strain was inoculated into PK-15 cells, the subcellular location of ORF6 was detected by IFA using ORF6 polyclonal antibody, the results showed that ORF6 was mainly distributed in the cytoplasm. We constructed eukaryotic expression plasmid p CAGGS-2ORF6. The expressioni of ORF6 protein in transfected HEK-293 T cells was detected by western blot.4. Construction of infectious clones and virus rescue in vitro Complete genome of PCV2 WUH strain was amplified by PCR with specific enzyme sites HindⅢand SacⅡ. Two copies genomes were ligated in tandem and inserted into the vector p Bluescript SK resulting into the infectious clone of wild type PCV2(w-2PCV2). At the same time, in order to construct the mutant strain deleting ORF6, The ATG sequence of ORF6 promoter it was mutated to ACG to silence ORF6 expression, which can not change the aa sequences of ORF2. Then we constructed the ORF6–deficient infectious clone(m-2PCV2) using the above methods. The wild type and mutant type infectious clones were transfected into PK-15 cells and infected blindly 3 passages. The wild type and mutant type infectious clones were shown to be infectious in vitroby using fluorescence quantitative PCR, the transcriptional detection of ORF2 m RNA, and IFA. We detected the proliferation of different viruses in vitro by Fluorescence quantitative PCR, the results showed that the virus was still able to replicate when ORF6 was deleted. It is confirmed that ORF6 is not the essential gene for viral replication. One step growth curve showed that the viral titers of the mutant virus were decreased significantly compared to the wild type virus. It revealed that ORF6 may affect the replication titer of PCV2.5. Biological function of PCV2 ORF6 The effects of PCV2 ORF6 on the cell apoptosis were investigated. We detected the activity of Caspase-3, Caspase-8, caspase-9 in PK-15 cells overexpressing ORF6 or infected by the virus. The results showed that ORF6 has little effect on the activities of Caspase-3, Caspase-8, caspase-9. However, ORF6-deficient mutant virus can significantly up-regulate the activity of caspase 3 and down-regulate the activity of caspase 8 The influence of apoptosis by ORF6 will be further studied. The eukaryotic expression plasmid p CAGGS-2ORF6 was transfected into PK-15 cells, then the cytokines were detected by fluorescence quantitative PCR. Cytokine species were IFN-β, TNF-α, RANTES, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p40 and IL-13. The results showed that ORF6 could increase the expression of IFN-β, TNF-α,IL-1β, IL-10, IL-12p40 and IL-13. At the same time, the wild type or mutant virus was inoculated in PK-15 cells, and the above cytokines were also detected. The results showed that the expression of IFN-β, TNF-α, IL-1β, IL-4, IL-6, IL-10, IL-12p40 and IL-13 induced by the mutant virus with ORF6 deletion was lower than that of the wild type virus.. Together with the results of cytokine induced by ORF6 expression and virus infection, It is evident that ORF6 could increase the expression of IFN-β, TNF-α, IL-1β, IL-10, IL-12p40 and IL-13.6. Effect of PCV2 ORF6 on IL-10 IL-10 is involved in the immune regulation and immune suppression induced by virus infection.We studieded the signaling pathway of ORF6 induced IL-10 expression by different signaling pathway inhibitors, it is observed that GF-109203 X, a specific inhibitor of protein kinase C, could significantly inhibit the expression of IL-10 induced by ORF6. Furthermore, the cells were treated by different concentrations of GF-109203 X inhibitor. The expression of IL-10 induced by ORF6 was inversely related to the inhibitor concentration, that is, the higher the inhibitor concentration, the lower the expression of IL-10 induced by ORF6. It revealed that ORF6 increased the expressionof IL-10 may through the protein kinase C pathway.