Cloning、Expression And Establishment Of Stable Expression Transfected PK-15Cells Line Of Procine TLR3Gene

The innate immune response was the frist line of host defense pathogen infection.Host cells recognized the pathogen-associated molecular patterns(PAMPs) through patternrecognition receptors（PRRs）which widely expressed on immune cell. Antiviral responsessuch as type I interferon (IFN) and cytokine production were successively induced invirus-infected animals. TLRs played a important roles in host immune action. Toll-likereceptor3(TLR3) was a PRRs known to initiate an innate immune response whenstimulated by virus double-stranded RNA (dsRNA). There are less reports of TLR3oflivestock at domestic and overseas. In our research, TLR3gene of Chinese Hezuo pigwas cloned, expressed and analyzed on the structure, evolutionay relationships, as well asbiological function. We established PK-15cells line which stably expressed greenfluorescent protein and TLR3gene as a cell model for pTLR3’ structure and function,which lay the foundation for further antiviral molecular mechanism study. Thefollowing are our research results:1.In this study, porcine Toll-like receptor3(pTLR3) gene and splicesomes ofpTLR3a、pTLR3b were cloned from porcine peripheral blood mononuclear cells (PBMCs)by reverse transcription-polymerase chain reaction (RT-PCR). The sequence analysisshows that the pTLR3open reading frame encode a deduced protein with905amino acidsresidues. The deduced amino acids sequence analysis indicate that it is a typical type Itransmembrane protein with a LRR ectodomain and a TIR cytoplasmic domains, theconserved structural features of TLRs.Due to the absence of extron3, the splicesomes ofpTLR3a、pTLR3b cann‘t encode the transmembrane and cytoplasmic domains. ThepTLR3a defected the whole extron3and pTLR3b defected C terminal parts of it. Thesimilarity of nucleotide sequence of pTLR3with those of cattle, equine, cat and human ismuch higher than as of rabbit and mice, and the lowest with the gallus‘.2.the extracellular domain of pTLR3was cloned into the prokaryotic expressionvector and constructed plasmid pGEX-4T-1-pTLR3, it was transformed into BL21toexpress fusion protein GST-pTLR3under the induction of42℃and0.6mmol/uL IPTG.Moreover, the GST-pTLR3fusion proteins then were purified, and the antiserum was madeby the immunized rabbits. Furthermore, the specificity of the antibody was to be identified by western blot and immunofluorescence. The results indicated that the antibodycombined specifically with the expressed protein of pTLR3in eukaryotic cell. Therefore, itwas showed that the pTLR3extracellular domain protein was highly expressed in BL21,and the antiserum with high titers and specificities was prepared.3.The pTLR3gene cloned into the Eukaryotic Expression vector and constructedplasmid pEGFP-N1-pTLR3and pcDNA3.1-His-pTLR3. the recombinant plasmidpEGFP-N1-pTLR3was transformed into DH5and conformed to express fusion proteinEGFP-pTLR3. Moreover, the PK-15cell line was transfected with the recombinantplasmid pEGFP-N1-pTLR3via FuGENE HD, and anti G418cell clones were screenedusing G418, both G418and GFP positive cell clones were continually cultivated. the geneexpression were confirmed by RT-PCR and Western Blot. The above results showed thatthe PK-15cell lines expressing pTLR3gene were established, and it provides a goodplatform for further investigation in pTLR3function.