Cloning and Bioinformatics Analysis of Toll-like Receptor 4 cDNA Full Length of Buffalo

The study aimed to clone Toll-like receptor 4(TLR4) gene of buffalo and analysis the sequence of this gene.Buffalo TLR4 gene was cloned by RT-PCR,the PCR products were sequenced and cloned into pMD20-T vector.Nucleotidesequence of this gene and structure of its encoding protein were predicted by using bioinformatics software.Sequence analysis results showed that the TLR4 gene ORF was 2526 bp in length and encoded 841 amino acids,including a signal peptide of 25 amino acids(aa) at the N terminal.Relative molecular weight of the encoding protein was 95.98 ku,and the isoelectric point(pI) was 6.37.Homology analysis showed that the nucleotide sequence of cloned gene shared 99.01% identities with the published sequence of buffalo TLR4 gene(DQ857349) in GenBank.The TLR4 gene sequence had a high homology compared with that of cattle,sheep,pig,horse,human and chimpanzee,which were over 80%,the homology with mouse and dog were 72.17% and 61.30% respectively,and that with chicken was only 53.94%.The protein structure prediction showed that TLR4 was a transmembrane protein composed of extracellular(1 to 634 aa),transmembrane(635 to 657 aa) and the intracellular rejoins(658 to 841 aa),which contained twelve LRR(leucine-rich repeats) tandem repeat ectodomains and a TIR(Toll-interleukin1-resistance) cytoplsmic domain.TLR4 of buffalo had been successfully cloned in the study,it lay a foundation for further researching the function of the gene and the protein characteristics.