水牛TLR4 cDNA全长的克隆及其生物信息学分析

本研究利用RT-PCR方法分段克隆水牛TLR4 cDNA后拼接成全长,并克隆于pMD20-T载体中,同时运用生物信息学软件对其核苷酸序列及编码蛋白质的结构进行分析和预测。结果表明,克隆的TLR4 cDNA ORF全长2526 bp,共编码841个氨基酸,N端具有由25个氨基酸组成的信号肽,该蛋白预测的分子质量为95.98 ku,等电点为6.37;水牛TLR4与GenBank中登录的水牛TLR4(DQ857349)核苷酸序列同源性达99.01%,与黄牛、绵羊、野猪、马、人和黑猩猩的同源性超过80%,与小鼠和狗的同源性次之,分别为72.17%和61.30%,与鸡的最低,仅为53.94%;该蛋白是由胞外区(1-634位氨基酸)、跨膜区(635-657位氨基酸)和胞内区(658-841位氨基酸)3部分构成的跨膜蛋白,胞外区含有12个LRR串联重复结构域、胞内区具有TIR结构域。本试验成功克隆了水牛TLR4 cDNA全长,并获得了核苷酸及其蛋白质的生物信息学分析数据,为进一步研究其功能奠定了基础。

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. Nucleotide sequence 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.