| Porcine circovirus diseases is a newly found contagion diseases, it caused by porcine circovirus type2(PCV2) which recognized to be a causative agent for postweaning multi-systemic wasting syndrome (PMWS). Science it first reported in Canada,1991, porcine circovirus diseases has become a highly important viral pathogen and bring a huge economic loss for the swine industry worldwide during last decades. PCV2could cause not only PMWS, but a series of diseases such as porcine dermatitis and nephropathy syndrome(PDNS), proliferative and necrotizing pneumonia(PNP), porcine reproductive and respiratory syndrome(PRRS).Calcium-binding protein(S100) belong to a small protein family. Members of this protein family have been shown to combine with Ca2+and interact with several effector proteins within cells thereby regulating expression of transcription factor, cell growth and differentiation, enzyme activities, inflammatory response and so on. S100is the key during Ca2+execute physical function. The molecular weight of porcine calcium-binding protein S100A9and S100A12are18.4kD and11.8kD. In our early lab work, we found PCV2could upregulated the expression of these2genes. There are reports that human S100A8/S100A9complex could enlarge the signal of promote inflammatory factor released by macrophagocyte through NF-κB pathway, and S100A12could also induce the production of cytokine and adhesion molecule through the same pathway. To the opposite, murine S100A9could inhibit NF-κB pathway activation. NF-κB is very important involve in anti-virus and anti-infection. This study aim to find the effect of porcine S100A9and S100A12in PCV2replication and NF-κB activation, results as follow:1. Gene clone and polyclonal antibody manufacture of porcine S100A9and S100A12the CDS of porcine S100A9and S100A12gene were cloned, and2prokaryotic expression vectors pGEX-KG-S100A9and pGEX-KG-S100A12were constructed. Use BL21strain to express protien and make mouse anti S100A9and S100A12polycloned antibody.2. Eukaryotic expression and subcellular localization analysis of porcine S100A9and S100A12Constructed2eukaryotic expression vector pEGFP-C1-S100A9and pEGFP-C1-S100A12, and transfected them into PK-15, Western Blot was used to confirm their protein expression. Confocal immunofluorescence analysis for subcellular localization revealed S100A9located in cytoplasm only and S100A12existed both in nucleus and cytoplasm.3. Effect of porcine S100A9and S100A12to the expression of IκB and IL-6, IL-8and IL-10in PK-15We found porcine S100A9and S100A12both could inhibit the protein expression of IκB through Western Blot. Real-Time PCR analysis showed the transcription of IL-6, IL-8and IL-10also been inhibited. Consider IκB and IL-6, IL-8and IL-10are upstream and downstream factors in NF-κB pathway, this suggest porcine S100A9and S100A12could inhibit NF-κB activation, and S100A12show a stronger effect.4. The influence of PCV2infection on the expression of porcine S100A9and S100A12geneAfter PCV2infection, the mRNA levels of porcine S100A9and S100A12significantly increased (p<0.05or p<0.01), along the time, the mRNA levels decreased but had another increase in48h. This suggest PCV2could increase the expression of porcine S100A9and S100A12and have a time correlative.5. The influence of porcine S100A9and S100A12on PCV2replicationTransfected pEGFP-C1-S100A9and pEGFP-C1-S100A12into PK-15, after24h observed the expression of aim gene by fluorescence microscope, then inoculated PCV2into cells. Using absolute quantitative, we found that the expression of S100A9and S100A12promote PCV2replication and have a significant difference inl2h (p<0.01). The PCV2copies in pEGFP-C1-S100A9group were1.76,1.12and1.54times than control group in12,24and36hours. The influence of S100A9weak in24and36h, but S100A12has a stable promotion. The PCV2copies in pEGFP-C1-S100A12group were1.51,3.99and3.2times than control group in12,24and36hours. These data showed S100A12may have a important role during PCV2infection and replication. |
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