Studies On The Potential Role Of Porcine C1QBP In PRRSV-induced Inflammation Response

Porcine reproductive and respiratory syndrome?PRRS?is an acute,highly-contact viral disease caused by porcine reproductive and respiratory syndrome virus?PRRSV?.PRRSV infection can lead to severe interstitial pneumonia,vasculitis,myocarditis and encephalitis.The typical symptoms are reproductive disorders in pregnant sows and dyspnea and growth retardation in piglets.It is reported that a large number of inflammatory cytokines can be induced at the site of PRRSV infection such as IL-6,IL-8,TNF-?,etc.In addition,some proteins are released to participate in the immune and inflammatory response,suggesting that the inflammatory response plays an important role in PRRSV infection.It is shown that the secretion of high mobility group box protein B1?HMGB1?is promoted in PRRSV-infected PAM and MARC-145 cells to enhance the inflammatory response induced by PRRSV,which also suggests that some secreted proteins are closely involved in PRRSV-mediated immune responses.C1QBP?complement component 1,q subcomponent binding protein?is a complement molecule C1q binding protein which can bind to the globular head domain of C1q.It is expressed in a variety of tissues and cell types such as epithelial cells,lymphocytes,dendritic cells,and platelets.C1QBP was originally discovered to be localized in mitochondria matrix,and was subsequently found to be localized in the cell membrane,nucleus,and the extracellular fluids.Moreover,C1QBP is a multi-ligand binding protein which can bind to pathogenic microbe-associated proteins,cellular proteins,and plasma proteins,and is widely involved in inflammation and infection processes.However,the role of C1QBP is unclear in PRRSV-induced inflammatory response.Therefore,we examined the effect of prokaryotic/eukaryotic expression of porcine C1QBP protein on PRRSV-induced inflammatory cytokine production.The specific research contents are as follows:1.Sequence analysis of porcine C1QBPThe amino acid sequences of C1QBP from different species were collected and a phylogenetic tree was constructed for genetic evolutionary analysis.The sequence alignment demonstrated that the porcine C1QBP is 93.6%identical to that of mouse,and83.3%identical to that of the human,respectively.The phylogenetic analysis indicated that the porcine C1QBP is evolutionarily located on the same branch with mouse C1QBP and belongs to an independent branch.2.Prokaryotic expression and purification of porcine C1QBPThe amplified porcine C1QBP was cloned into pET-28a to generate the pokaryotic expression construct pET-28a-C1QBP.Then the pET-28a-C1QBP plasmid was transformed into BL21 Escherichia coli to express C1QBP protein with IPTG.We obtained a specific C1QBP protein appeared at 35kDa in SDS-PAGE,which was mainly expressed in soluble form and purified by nickel column.The final protein concentration was 0.5 mg/mL.The endotoxin of purified C1QBP was reduced by a endotoxin removal kit that removed 50%of endotoxin in recombinant C1QBP.Subsequently,the cytotoxicity of the protein was detected by MTT assay.It showed that the concentration of C1QBP protein below 80?g/mL is not harmful for cells.3.Purified C1QBP recombinant protein induces inflammatory cytokines expressionThe purified porcine C1QBP protein from Ecoli was added to PAM cells.The mRNA levels of IL-1?,IL-6,IL-8,TNF-?and RANTES were detected by real-time PCR.The results showed C1QBP treatment can promote the expression of inflammatory cytokines.And furthermore,Western blot and IFA assays showed that the purified C1QBP protein can promote the phosphorylation level of p65 and its nuclear translocation.These results indicated that the C1QBP protein plays an important role in pro-inflammatory cytokines production.4.Knockdown of C1QBP inhibits LPS/PRRSV-induced inflammatory responsePAM cells were transfected with C1QBP-specific siRNAs,and then were treated with LPS or infected with PRRSV to induce the production of inflammatory cytokines.We analysed the effect of si-C1QBP on LPS/PRRSV-induced inflammatory cytokines with real-time PCR.It showed that knockdown of C1QBP inhibited LPS/PRRSV-induced inflammatory cytokines expression.5.PRRSV infection promotes the secretion of C1QBP and does not alter its mitochondrial localizationBy analyzing the secretome information of PRRSV-infected MARC-145,we found that the secretion of C1QBP was significantly up-regulated after PRRSV infection.On MARC-145 and PAM cells,cell lysate and cell culture supernatant was collected at different time points after PRRSV infection.Westrn blot was used to detect the expression of C1QBP in the cells and supernatant.The results showed that the intracellular C1QBP was not almost changed after infection,while the C1QBP in cell culture supernatant was significantly increased.It indicated that PRRSV infection absolutely promoted the secretion of C1QBP.At the same time,IFA assay revealed that PRRSV did not alter the mitochondrial localization of C1QBP.6.Purified C1QBP enhances PRRSV-induced inflammatory responses.PRRSV-infected MARC-145 and PK-15^CD163 cells were treated with purified C1QBP protein from Ecoli,and the transcription level of inflammatory cytokines was detected by real-time PCR.We found that C1QBP can enhance the expression of inflammatory cytokines induced by PRRSV.In addition,it is showed that C1QBP can enhance PRRSV-induced NF-?B activation and phosphorylation levels of p65 through the NF-?B luciferase reporter system and Western blot assay.At the same time,C1QBP protein purified from mammalian cells HEK-293T and insect cell Sf9 can enhance PRRSV-induced inflammatory cytokines expression on PRRSV-infected PAM cells.