Characterization Of Woodchuck Innate Immunity Related Molecules RelA, ISG15and Preliminary Study Of TLRs Signaling In Woodchuck Cell Line

ObjectiveWoodchuck model has been widely used in the pathogenesis study and the therapeuticstrategies evaluation for hepatitis B virus (HBV) infection. Innate immune response toHBV infection and the application of TLR agonists as a therapeutic strategy is anarising hotspot in HBV study. However, the sequences of woodchuck innate immunemolecules RelA, ISG15are still unkown. In this study, we characterized woodchuckRelA, ISG15and the reference molecule GAPDH, and explored the expressing profileof TLR signaling related molecules in woodchuck fibroblast cell line W12/6under thestimulating of TLR1/TLR2, TLR3, TLR4agonists.Method1. Consered primers were designed by alignment of mammalian RelA, ISG15, andGAPDH sequences published in Genebank. Total RNA was extracted fromwoodchuck W12/6cells, and cDNA sequences of woodchuck RelA, ISG15,GAPDH were amplified by RT-PCR using the respective conserved primers. ThePCR products were cloned into pMD18-T vectors, the plasmids harboring RelA,ISG15, and GAPDH were identified by PCR screening, endonuclease digestionand sequencing. The obtained sequences of woodchuck RelA, ISG15, GAPDHwere analyzed. 2. W12/6cells were stimulated with TLR1/TLR2, TLR3, TLR4agonists and totalRNA was extracted at3hours,6hours, and12hours after the stimulation. Theexpressing profile of TLR signaling related molecules IRF1, IRF3, IRF5, IRF7,IFNβ, IL1β, IL6, ISG15, and RelA was analyzed by Realtime-PCR.Results1. The partial sequence of woodchuck RelA (1559base pairs (bp)) was obtained, thepredicted protein include493amino acid (aa) residues. The sequence ofwoodchuck RelA shows high homology with the RelA of other species, rangingfrom87%to89%at the nucleotide (nt) level and88%to90%at the aa level.2. The partial sequence of woodchuck ISG15(370bp) was obtained, the predictedprotein include123aa residues. The sequence of woodchuck ISG15shows highhomology with the ISG15of other species ranging from74%to78%at the ntlevel and57%-69%at the aa level.3. The complete coding sequence (CDS) of woodchuck GAPDH (1214bp) wasobtained, the predicted protein includes334aa residues. The sequence ofwoodchuck GAPDH shows high homology with GAPDH of other species rangingfrom87%to93%at the nt level and95%to98%at the aa level.4. The expressing profiles of IRF1, IRF3, IRF5, IRF7, IFNβ, IL1β, IL6, ISG15,RelA in woodchuck fibroblast cell line W12/6under the stimulating of TLR1/2,TLR3, TLR4agonists were as below:(1) After the stimulation with TLR1/2agonist, only the expression of IL1β wasslightly increased at3hours, while the expression of all molecules, except IL1βand IL6were up regulated at6hours; After that the expression of all investigatedmolecules fell back to the base line at12hours.(2) After the stimulation with TLR3agonist, the expression of all molecules, exceptIRF3, IRF5, IL1β, ISG15were up regulated at3hours, while the expression ofall investigated molecules fell back to the base line at6hours. And at12hours, only the expression of IRF5was up regulated.(3) After the stimulation with TLR4agonist, the expression of all molecules were upregulated except IRF3and IRF5at3hours, while only the expression of IRF1andIL6were up regulated at6hours. However, the expression of IRF1, IRF5, IRF7,IL1β, IL6, ISG15and RelA were up regulated again at12hours.Conclusion1. The cDNA sequences of woodchuck RelA, ISG15are obtained, providing thebasis for the application of woodchuck model in innate immune related study ofHBV infection.2. Woodchuck RelA, ISG15have high identities with their counterpart of human,similar to our previous studies on woodchuck IFNAR, IL-6and IL-15.3. TLR signaling related molecules were up regulated in W12/6cells under thestimulation of TLR1/2, TLR3, and TLR4agonists, with a time-dependent manner.