Immunosuppressive Effect Of IL-10 On The Immune Response Induced By PRRSV Or CSFV Vaccine

Porcine Reproductive and Respiratory Syndrome?PRRS?and Classical swine fever?CSF?are both important swine infectious diseases.After PRRSV infects pigs,it will cause immune suppression and affect the production of CSFV antibody.Therefore,PRRSV not only causes huge economic losses for pig breeding industry,but also brings difficulties for clinical prevention and control.Previous studies have shown that both PRRSV and CSFV will cause a series of immune-related cytokine levels to change after infection in pigs,and finally play a role in regulating the body's immune response and playing an antiviral effect.Among them,IL-10,as a typical immunosuppressive factor,has an important impact on the body's normal immune response.Studies have found that both PRRSV and CSFV can induce increased IL-10 secretion in pigs infected with them,but the significance of the induced high expression of IL-10 on the immunosuppressive effect of PRRSV is still unclear.In order to further explain the root cause of PRRSV induced immunosuppression and reveal the molecular significance of IL-10 mediated immunosuppression of PRRSV,and to provide a solution for prevention and control of PRRSV,In this study,we used human recombinant adenovirus expression system and a technique called RNA interference to construct two recombinant adenoviruses that express porcine IL-10 and the si RNA that specifically silences porcine IL-10,respectively.Then,with recombinant adenovirus infection cells and the body to regulate the levels of IL-10,we examined the effect of over-expression of IL-10 on the proliferation of PRRSV and CSFV,and also on serum antibody production in piglets after immunization with PRRSV or CSFV vaccine.The main test content and test results are as follows:?1?Construct r HAd-IL10 and r HAd-si IL10 recombinant adenovirusThe complete pig IL10 gene was obtained by PCR,and sh RNA double-stranded DNA molecules that could inhibit the expression of pig IL-10 were designed and synthesized.The recombinant adenovirus plasmids of p Ad-IL10 and p Ad-si IL10 were obtained by inserting them into human adenovirus vectors respectively.We identified the correct recombinant adenovirus plasmids and then transfected them into HEK-293A cells with Pac I linearized for recombinant adenovirus packaging,and finally obtained r HAd-IL10 and r HAd-si IL10recombinant adenovirus with TCID₅₀ of 10^-10.25/m L and 10^-9.65/m L,respectively.?2?Verify the expression activity of r HAd-IL10 and r HAd-si IL10 recombinant adenovirusIn the experiment,RT-q PCR and Western Blot were used to detect the activity of the target factors of the two recombinant adenovirus expressions.The test results showed that the infection of 10 MOI r HAD-IL10 on 3D4/21 cells could significantly inhibit the induction of LPS on the expression of IL-12 in 3D4/21 cells?P<0.01?,and the expression level of IL-12m RNA was decreased by about 40%.In addition,the expression of IL-10 was inhibited after3D4/21 cells were infected with 1 MOI and 10 MOI r HAd-si IL10,the expression of IL-10m RNA was down-regulated by 25%and 40%,and the expression of IL-10 by Western Blot was down-regulated by 10%and 40%,respectively.It was proved that both IL-10 and si RNA expressed by r HAD-IL10 and r HAd-si IL10 had the expected biological activity,which laid a foundation for the follow-up study.?3?Effect of over-expression of IL-10 on intracellular proliferation of PRRSV and CSFVIn this study,r HAD-IL10 and r HAd-si IL10 were used to infect cells to realize the up-regulation and down-regulation of IL-10 expression.In combination with fluorescence microscope observation,RT-q PCR,Western Blot and TCID₅₀ assay,it was found that compared with the control group infected with r HA-EGFP,the m RNA expression of PRRSV SD16-GFP virus increased by 1 times as over-expression of IL-10 in PAM cells after infection24 h.After PRRSV SD16-GFP virus infected Marc-145 cells,increased expression of GPF was observed by fluorescence microscopy after infection 48 h and 60 h,and the expression levels of PRRSV m RNA and N protein were respectively about 2.3 and 1.2 times of those in the control group after infection 48 h,and virus TCID₅₀ increased significantly.Moreover,after over-expression of IL-10,m RNA expression levels of CSF vaccine virus were detected after infection PK-15 and 3D4/21 cells 48 h with CSF vaccine virus,which were about 3 and2.5 times that of the control group,respectively.However,m RNA expression levels of CSF vaccine virus were about 25%and 30%lower when IL-10 expression was down-regulated.The results showed that the over-expression of IL-10 promoted the proliferation of PRRSV SD16-GFP virus and CSF vaccine virus in cells,and the down-regulation of IL-10 expression significantly inhibited the replication of CSF vaccine virus in PK-15 and 3D4/21 cells.?4?Effects of over-expression of IL-10 on serum antibody production in piglets immunized with PRRSV or CSFV vaccineThrough Infection piglets aged 3-4 weeks with r HAd-IL10 or r HAd-si IL10 recombinant adenovirus by injecting the virus into the muscle behind the ear,we can regulate the expression of IL-10 in the body.The test found that compared with the control group infected with r HAd-EGFP,the production of serum antibody was significantly inhibited and the antibody positive rate of piglets significantly decreased in the over-expressed IL-10 group after immunization with PRRSV or CSFV vaccine.In addition,the antibody change trend of piglets in the group with down-regulated IL-10 expression was similar to the control group,showing an overall growth trend,but the PRRSV antibody level of the group with inhibited IL-10 expression showed a rapid growth trend earlier than the control group.This confirmed the significance of high expression of IL-10 in PRRSV immunosuppression.In conclusion,we build r HAd-IL10 and r HAd-si IL10 recombinant adenovirus through the experiment,and confirmed that the over-expression of IL-10 can promote PRRSV SD16-GFP and CSF vaccine virus proliferation in cells,but down-regulation of IL-10 expression inhibited the replication of CSF vaccine virus in PK-15 and 3D4/21 cells.IL-10 promotes virus proliferation,which may be the cause of PRRSV's persistent infection,it remains to be further research.In addition,after the over-expression of IL-10 in piglets,the expression of PRRSV serum antibody was inhibited in the vaccinated piglets,but when the expression of IL-10 was inhibited by si RNA,the antibody level of PRRSV showed a rapid increase earlier than that of the control group.This finding indicates that IL-10 mediates the immunosuppression caused by PRRSV,which provides a basis for the development of vaccine and the prevention and control of immunosuppression.