Cloning Of The Porcine Retinoblastoma-Inhibiting Gene â…  And Its Effect On The Induction Of Type â…  Interferon

The innate immune systems is essential to protect the host From the virus infection.They play important roles in the innate immunity by recognizing pathogen-associated molecular patterns(PAMPs).PAMPs trigger the innate immune system to secrete the antiviral cytokines IFN-b and IFN-a as well as cytokines that induce an inflammatory response.Toll-like receptors(TLRs) constitute a widely studied family of PRRs,playing an important role in detecting RNA virus.TLR3 allows cells to detect dsRNA,TLR7 and TLR8 allow cells to detect ssRNA.Toll-like receptors(TLRs) were identified as transmembrane signal transduction proteins,Viral RNA is recognized in the endosome by membrane-bound Toll-like receptors,they can not sense the presence of dsRNA in the cell cytoplasm RIG-I-like receptor is a recent discovery of pattern recognition receptor and for RNA-sensing in the cytoplasm,RIG-I was recently shown to detect,which is an essential transcription factor regulating the expression of typeⅠIFN and other cytokine genes during virus infection.The research on hunman RIG-I is clear.In the present study,porcine RIG-I molecule was cloned and its function involving in typeⅠIFN signal pathway was characterized. The most research works were as following:1.Cloning and sequence analysis of porcine RIG-I geneThe complete cDNA encoding RIG-I was amplified by one-tube reverse transcription polymerase chain reaction(RT-PCR).The cDNA fragment was further cloned and the complete sequences were determined.The results indicate that the open reading frame of porcine RIG-I is consisted of 2 832 bp which encodes 943 amino acid residues.Compared with the porcine RIG-I gene,cattle,horse human,monkey, chimpanzee,it was found that there was 82.3%,80.2%,80.2%,79.7%and 78.8%aa identity respectively.The analysis of the phylogenetic tree also showed that porcine RIG-I gene is more closely related to the cattle than it is to the horse.2.Construction of porcine RIG-I recombinant plasmidsThe cDNA encoding the complete RIG-I,N-terminal domain of RIG-I,C-terminal domain of RIG-I was isolated by PCR from pMD18-RIG-I and cloned into the pCMV-Tag-2B with BamHⅠand HindⅢdigestion,which named Tag-RIG-I, Tag-RIG-I-N,Tag-RIG-I-C.The recombinant plasmids were verified by digestion analysis.3.Function study on induction of typeⅠinterferon of Porcine Retinoblastoma-inhibiting gene IPorcine kidney(PK-15) cells transfected with Tag-RIG-I,Tag-RIG-I-N, Tag-RIG-I-C,from luciferase reporter,the results indicate that Tag-RIG-I and Tag-RIG-I-N could induce IFN-β,and activated IRF3 and NF-κB.Deletion mutant analyses further revealed that over expression of CARD alone activated the downstream signaling and inhibited poly(I:C)-mediated production of IFN-β.Moreover,the mutant of RIG-I lacking CARD was not capable of transmitting signals.