PCV3 Cap And Rep Protein Induce Related Cytokine MRNA Transcription And IL-10 Expression Mechanism

Porcine circovirus type 3(PCV3)is a new type of circovirus discovered in 2016 and is circulating in many countries worldwide.It is currently believed that PCV3 infection is mainly related to clinical symptoms such as reproductive disorders and dermatitis nephrotic syndrome.The emergence of PCV3 has continuously escalated the impact of pathogenic PCVs on the pig industry,but the pathogenesis of PCV3 is still unclear.In this study,passageable porcine alveolar macrophages(3D4 / 21)were selected.From the perspective of PCV3 Cap and Rep proteins,the eukaryotic expression vectors of PCV3 Cap and Rep were constructed to investigate their regulation of 3D4/ 21-related cytokine expression At the same time,the effect on the expression level of immunosuppressive factor IL-10 was clarified,and then related pathway inhibitors were used to explore the relevant pathway information regulating IL-10 expression,providing a basis for clarifying the pathogenicity of PCV3.1.The expression of the eukaryotic expression vector of PCV3 major encoding proteins in 3D4/21 and Vero cellsThe expression and verification of PCV3 Cap and Rep proteins in 3D4 / 21 and Vero cells found that PCV3 Cap and Rep can be expressed in porcine alveolar macrophages(3D4 / 21)and Vero cells,in which PCV3 Cap and PCV2 Cap proteins are similar.48 hours after transfection of 3D4 / 21 cells,they were mainly localized in the nucleus.The functional correct expression of PCV3 Cap in Vero cells was verified by PCV3 positive pig serum,and 48 PCV3 negative sera were screened by Vero cell IFA method expressing PCV3 Cap,and used for the establishment of PCV3 Cap ELISA detection antibody system and threshold determination.This method can be used for detection and identification of porcine serum PCV3 antibodies.2.PCV3 Cap and Rep proteins affect 3D4 / 21 cell-related cytokine expressionThe effect of PCV3 Cap and Rep on the expression of porcine alveolar macrophages(3D4 / 21)-related cytokines was explored by q PCR.The results showed that transfection of PCV3 Cap and Rep eukaryotic recombinant plasmids into porcine alveolar macrophages would lead to cytokine secretion.disorder.Both PCV3 Cap and Rep can significantly induce IFN-? and IFN-? up-regulation,which may be related to early antiviral infection,significantly up-regulate the m RNA levels of IL-6 and TNF-?,and be related to triggering the body's inflammatory response.PCV3 Cap and Rep showed opposite effects in inducing IFN-? expression,PCV3 Rep up-regulated IFN-? expression,and PCV3 Cap did the opposite.In addition,PCV3 Cap significantly increased the expression and secretion of IL-10 in 3D4 / 21 cells 24 h after transfection.Compared with PCV3 Rep,PCV3 Cap is the main PCV3-encoded protein that up-regulates IL-10 expression.3.PCV3 Cap up-regulates IL-10 expression in 3D4 / 21 cells through the NF-?B pathwayFurther research found that IL-10 m RNA levels increased significantly with the increase of PCV3 Cap expression(P <0.01).And through inhibitor experiments,it was found that,similar to PCV2 Cap,NF-?B signaling pathway is involved in the process of PCV3 Cap inducing IL-10 upregulation.It is suggested that PCV3 infection can play a role in upregulating IL-10 expression through its Cap activating NF-?B pathway,and the result will provide a mechanism for elucidating the mechanism of PCV3-infected pig body regulating IL-10 and other cytokines and immunosuppressive regulation Theoretical basis.