Man Toll-like Receptor, The Extracellular Domain Of Gene Cloning And Sequence Analysis

Toll-like receptors(TLRs) were discovered in Drosophila melanogaster first.In 1997,the TLR4 of hunman that like the protein Toll of Drosophila melanogaster was discovered.Eleven human TLRs have been identified untill now.TLRs belong to IL-1R superfamily because their cytoplasmatic domains that participate in recognizing a variety of microbial molecules are so similar to each other.TLRs take part in innate immunity and adaptive immunity by recognizing many kinds of conserved pathogenic structures termed pathogen-associated molecular patterns(PAMPs).In the eleven discovered TLRs,we know less about TLR1 than the other tens.TLR1 occur in various immunocyte such as monocyte,neutrophile,T lymphocyte,B lymphocyte,natural killer cell and so on.TLR1 recognize triacylated lipoproteins in many kinds of microorganism that can activate immune responses through TLR1- and TLR2-dependent pathways.TLR1 polymorphism could regulate the innate immune response and clinical susceptibility to a wide spectrum of pathogens to lipopeptides.But we know few about what kinds of microorganism TLR1 could discriminate,how the mechanism of its recognition and signal transduction pathways works,what diseases may be defended by the mechanism.At present,TLR2,TLR3,TLR4,TLR7 and TLR9 protein have been expressed one after another and applied in research of how the mechanism of receptor protein's recognition and signal transduction pathways work.There are few reports about the clone and expression of protein TLR1 invitro.Objective:To obtain the positive E.coli BL21 clones which could express the extraceUular regions of human TLR1 DNA stably,the genes of TLR1 were amplified and cloned into E.coli BL21.By this reseach,the basis will be provide for further study the machnism that how does the TLR1 recognize pathogen molecules and how does the signal pathway work,and the new idea of disease therapy may be found.Methods:According to the sequence of human TLR1 in GenBank, primers of Human TLR1 extracellular cDNA codon domain were designed.The objective gene fragment(1 735 bp) was amplified by RT-PCR and digested by Ncoâ… and Notâ… ,and then cloned into pET30a(+) plasmid.The constructed recombinant plasmid pET30a(+)-TLR1 was transfected into E.coli BL21 (DE3).Results:â‘ Human PBMC were isolated from peripheral venous blood provided by healthy donors with Ficoll-Hypaque density gradient centrifugation.RNA was extracted using the Trizol total RNA reagent.â‘¡The primers of Human TLR1 extracellular cDNA codon domain with Nocâ… and Notâ… were designed.The genes of TLR1 were amplified by RT-PCR and the amplified products were extracted with Gel Extraction Kit.â‘¢The DNA of extracellular regions of human TLR1 and that of prokaryotic expression vector pET30a(+) plasmid were ligated by T4 DNA Ligase.The constructed recombinant plasmid pET30a(+)-TLR1 was transfected into E.coli BL21(DE3) and the positive clones were identified by PCR.â‘£The positive clones were sequenced by Shanghai Sangon Biological Engineering Technology and Service Co.,Ltd,and the sequence was aligned in NCBI.The results showed that the inserted fragment(1 735 bp) and the sequence of TLR1 in Genebank shared 99%homology,which confirm that the clone sequence were correct.Conclusions.The genes of Human TLR1 extracellular cDNA codon domain were amplified successfully and the size of amplified product was 1735 bp.Moreover,the obtained gene fragment was also successfully cloned into prokaryotic expression vector plasmid pET30a(+),and the results by sequencing showed that the clone seuqunce of TLR1 were correct.