Preliminary Research On The Effect Of CD46 Molecule On Immunological Function Of Dendritic Cells Infected With Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus?BVDV?can infect a variety of animals,but mainly cattle.Because of reproductive obstacle and persistent infection,BVD causes a significant losses in cattle farming worldwide.CD46 is a specific membrane receptor for BVDV,which mediates the viral infection of bovine dendritic cells?DCs?,DCs are both immune response promoters and immune tolerance inducers.Therefore,in order to preliminarily investigate the possible role of CD46 molecule in immune responses mediated by dendritic cells infected with BVDV,the following experiments were carried out in this study:?1?The expression profile of CD46 gene in Xinjiang brown cattle different tissues — heart,liver,spleen,lung,kidney,small intestine,large intestine,stomach,muscle,ovary and breast—was analyzed by Real-Time PCR.The results showed that CD46 gene of Xinjiang Brown Cattle was expressed in all 11 samples,and the highest in the liver?P<0.05?.?2?In this experiment,specific primers for the bovine CD46 gene containing entire open reading frame?ORF?were designed according to the nucleotide sequence?NM₀01242561.1?published in GeneBank.The CD46 gene was amplified by RT-PCR from Xinjiang Brown Cattle spleen and cloned into pVAX1 vector,producing the recombination plasmid pVAX1-CD46.After bioinformatic analysis of the sequence data,the extracellular domain of CD46 gene without the signal peptide sequence was amplified and cloned into pET-28 a expression vectors.The recombination plasmid pET-28a-?CD46 was transformed into E.coli.Rosetta?DE3?,and expressed fusion protein His-?CD46 after induction by isopropyl-?-D-thiogalactoside?IPTG?.An anti-His-?CD46 polyclonal antibody was generated by immunizing subcutaneously New Zealand white rabbits with the His-?CD46 fusion protein purified by gel extraction,and the result of ELISA indicated that the titer of the antibody was 1:128000.BHK-21 cells were transiently transfected with eukaryotic expression plasmid pVAX1-CD46,and the specificity of the rabbit anti-His-?CD46 polyclonal antibody was assessed by Western blot.Results showed that the expression protein of pVAX1-CD46 in BHK-21 cells could be specifically recognized by the rabbit anti-His-?CD46 polyclonal antibody.?3?According to the siRNA design guidelines,three short hairpin oligonucleotides and complementary strands were designed to target the consensus sequences of bovine CD46 gene.The sense and antisense oligonucleotides were annealed,and cloned into the expression vector pLL3.7 containing the mouse U6 promoter.The resultant plasmids,which express short hairpin RNA?shRNA?against bovine CD46 gene,were transfected into MDBK cells for 48 hours using Lipofectamin 2000.Real-time PCR analysis showed that recombinant plasmid pLL3.7-CD46-shRNAc effectively knocked down CD46 mRNA by 58.67%?P < 0.05?.Western blot results also showed that the expression of CD46 protein was significantly inhibited in MDBK cells transfected with the recombinant plasmid pLL3.7-CD46-shRNAc.?4?The mononuclear cells isolated from peripheral blood of Xinjiang brown cattle were stimulated to differentiate into bovine monocyte-derived dendritic cells?MDDC?using brGM-CSF and brIL-4.MDDCs were transfected with the pVAX1-CD46 and pLL3.7-CD46-shRNA respectively,and followed by infectionwith BVDV,resulting in CD46 gene-modified dendritic cells.Real-time PCR was used to detect mRNA expression levels of cellular phenotype and cytokines in the CD46 gene-modified DCs infected with BVDV.The results showed that,compared with untransfected cells,the mRNA levels of CD40,CD80,CD86 and MHCII in CD46 gene overexpression MDDCs were significantly increased?P<0.05?;CD40mRNA levels in CD46 gene silencing MDDCs was also increased significantly?P<0.05?;CD86 mRNA level was not changed?P>0.05?;CD80 and MHCII mRNA levels were significantly decreased?P<0.05?.Compared with untransfected cells,the levels of IL-10 and IL-12 mRNA in CD46 gene overexpression group were not significantly changed?P>0.05?,while their mRNA expression levels were significantly decreased in CD46 gene silencing MDDCs?P<0.05?.In summary,this preliminary study revealed a new perspective on the effect of CD46 molecule on maturation of dendritic cells infected with BVDV and proliferation and differentiation of naive T cells,providing new ideas for addressing the immunosuppressive effects of DCs to BVDV,Which is of great significance to control the occurrence of BVD.