Molecular Cloning, Tissues Expression Profile On Pocine TLR7 And MyD88 And Study On Expression Of MyD88 In Pichia Pastoris

The innate immune system is the first line of the defensive mechanisms that protect hosts from invading microbial pathogens.Host cells express various pattern recognition receptors(PRRs) that sense diverse pathogen-associated molecular patterns(PAMPs),ranging from lipids, lipoproteins,proteins and nucleic acids.Recognition of PAMPs by PRRs activates intracellular signaling pathways that culminate in the induction of inflammatory cytokines,chemokines, interferons(IFNs) and upregulation of MHC-Ⅱand co-stimulatory molecules to influence the adaptive immune system.Toll-like receptors(TLRs) are one of the important PRRs families and are typeⅠmembrane proteins.To date,13 mouse TLRs and 11 human TLRs have been identified, and each TLR appears to recognize distinct PAMPs derived from various microorganisms, including bacteria,viruses,protozoa and fungi.As a member of Toll-like receptor(TLR) family,Toll-like receptor 7(TLR7) can identify the single strand RNA of virus and help the host to take corresponding control of immune response by inducing to some cytokines expression in animal body such as interferon(IFN)-αand interleukin(IL)-12.It was demonstrated that some viruses(Including vesicular stomatitis virus, influenza virus,Human immunodeficiency virus-Ⅰand Hepatitis C Virus etc) stimulate immune responses through TLR7.Myeloid differentiation primary response protein 88(MyD88) is an important adapter protein in the signal transduction pathway mediated by interleukin-1(IL-1) and Toll-like receptors.New evidence shows that MyD88 also participates in interferon-γ-induced cellular responses and has a role to prolong the half-time of some mRNA induced by interferon-γ.Here we cloned cDNA of TLR7 gene from total RNA of mesenteric lymph nodes(MLN) in Neijiang pig by RT-PCR,and RACE(rapid amplification of cDNA ends).Sequence analysis indicated that the porcine TLR7 cDNA cloned was 3834 nt in length(GenBank accession number EF469730) and the open reading frame encodes a deduced protein with 1050 amino acids residues. The analysis of deduced amino acids sequence indicated that TLR7 is a typical typeⅠtransmembrane protein with multi-LRR-RI ectodomains and a TIR cytoplasmic domains,the common structural features of TLRs.The comparison of the deduced amino acids sequence of porcine TLR7 with those of cattle,dog,human,cat and mouse showed that the amino acids homology were 90.8%,87.4%,84.9%,86.7%and 78.2%respectively and the TLR7 is highly conserved among the different mammal species.Using real-time quantitative PCR,we detected TLR7 mRNA expression in a panel of porcine tissues(heart,lung,spleen,liver,kidney,skeletal muscle,brain,jejunum,Peyer's patches and mesenteric lymph nodes) with the greatest levels of expression observed in mesenterie lymph nodes(MLN),and Peyer's patches.Pocine MyD88 cDNA was cloned using mRNA isolated from adult swine mesenteric lymph nodes(MLN).The sequences were confirmed in three mesenteric lymph nodes from various adult.Nucleotide sequencing of pocine MyD88 revealed a 897bp cDNA sequence.Including the pocine MyD88 structural gene.The nucleotide sequence of pocine MyD88 has been submitted to the GenBank nucleotide databases(GenBank accession number EF198416).A predicted open reading flame(ORF) lay between bp 45 and 926 and encoded a 293aa protein.To determine the structural domains in pocine MyD88.the amino acid sequence was analyzed using the SMART program.The deduced amino acid sequence of pocine MyD88 possesses a typical MyD88 domain including an N-terminal death domain.as well as interm ediate and C-terminal TIR domains.Two other regions were intrinsically disordered.The alignment of amino acid sequences for MyD88 showed that the pocine MyD88 sequence is highly conserved among sequences from other species.The amino acid sequence of the pocine MyD88 is 88.4%,85.7%,78.8%and 78.2% identical to that of human,cattle rat and of mouse MyD88,respectively.These results indicated that pocine MyD88 is more similar to cattle and human than to mouse MyD88 at aminoacid levels.Phylogenetic analysis showed that pocine MyD88 belonged to the group containing cattle MyD88 and human MyD88.In addition,pocine MyD88 was more closely related to human MyD88 and cattle MyD88 than to mouse MyD88 in terms of identity.Real-time PCR analysis shows that the MyD88 gene is expressed in various tissues,but at different levels.The expression levels of this gene are higher in Peyer's patches and mesenteric lymph nodes(MLN).The expression levels in musle and heart are very lower and aren't almost be detected.Pichia pastoris is one of the most used expression systems for the high level expression of heterologous proteins.This expression system has the advantages of Prokaryotic systems expression and Eukaryotic expression systems.①the simplicity of techniques needed for the molecular genetic manipulation of P.pastoris and their similarity to those of Saccharomyces cerevisiae,one of the most well-characterized experimental systems in modern biology;②the ability of P.pastoris to produce foreign proteins at high levels,either intracellularly or extracellularly;③the capability of performing many eukaryotic posttranslational modifications, such as glycosylation,disulfide bond formation and proteolytic processing;and④the availability of the expression system as a commercially available kit.In our studies,we used pPIC9K as expression vector and KM71 as host strains to express porcine MyD88 protein.After the porcine MyD88 gene with EcoRⅠand NotⅠrestriction Sites and His tag was cloned to pMD18-T vector, pMD18-T-MyD88 and pPIC9K were cutted by EcoRⅠand NotⅠand the cutted fragments were ligated by T4 DNA ligase.The porcine MyD88 gene expression vector,pPIC9K-MyD88,was constructed successfully after sequencing.The expression vector pPIC9K-MyD88 used for transformation,was linearized by SacⅠ.Pichia pastoris KM71 strains were made competent and transformed with SalⅠ-linearized pP9K-MyD88 by electroporation.After selection by MD plates, YPD plates containing G418,some positive colonies which exhibited different anti-G418-levels were obtained.We selected different anti-G418-levels positive clones and induced them to expression porcine MyD88 protein by methanol.The culture supernatant contained the target protein was purifed by affinity chromatagraphy used HisTrap^TM HP and BioLogic DuoFlow. Purified MyD88 expression products were analyzed by SDS-PAGE and found a major protein band at a molecular weight of 34 kDa,which is consistent with the molecular weight of pig MyD88.The porcine MyD88 was expressed successfully.