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目的原核表达树鼩(tree shrew,ts)白细胞介素-6(interleukin-6,IL-6)基因,并分析ts IL-6蛋白分子特征。方法以人、猴、啮齿类动物等IL-6 m RNA的共有序列作为查询序列,从树鼩c DNA数据库中进行本地blast,获得预测ts IL-6核苷酸序列,将其翻译成氨基酸序列,用MEGA 6软件进行核酸序列和氨基酸序列比对,绘制进化树。参考预测IL-6基因序列设计引物,PCR扩增IL-6基因序列,克隆至原核表达载体p ET-28a(+),构建重组表达质粒p ET-28a(+)-IL-6;转化感受态E.coli BL21(ED3),IPTG诱导表达,重组蛋白经14 KD透析膜梯度透析纯化;利用蛋白质在线数据库expasy进行蛋白质结构同源建模,并用Signal P sever预测ts IL-6的信号肽序列,经模板与预测模型的结构拟合来预测树鼩IL-6的gp130结合位点。结果 ts IL-6核苷酸和氨基酸序列与高等灵长类动物有较高的同源性;重组表达质粒p ET-28a(+)-IL-6经双酶切鉴定证明构建正确;重组表达蛋白相对分子质量约为24 000,纯化后可见单一目的条带,浓度为0.2 mg/ml;h IL-6和ts IL-6蛋白的空间结构较类似,ts IL-6的gp130结合位点Ⅱa位于A和C螺旋间,位点Ⅲa位于A和B螺旋的柔性链区及分子近C-端。结论 ts IL-6的核苷酸和氨基酸序列与高等灵长类动物具有较高同源性,成功构建的重组原核表达质粒p ET-28a(+)-IL-6,为后续IL-6相关的病理研究奠定了基础。
Objective To express the gene of interleukin-6 (IL-6) of tree shrew (ts) in prokaryotic cells and analyze the molecular characteristics of ts IL-6 protein. METHODS: Local IL-6 m RNA sequences from humans, monkeys, rodents and other rodents were used as query sequences. Local blast was performed from a tree DNA library to obtain predicted ts IL-6 nucleotide sequences and translated into amino acid sequences , Using MEGA 6 software for nucleic acid sequence and amino acid sequence alignment, mapping phylogenetic tree. The sequence of IL-6 gene was designed according to the predicted IL-6 gene sequence, and then cloned into the prokaryotic expression vector p ET-28a (+) to construct the recombinant expression plasmid p ET-28a (+) - IL- The E. coli BL21 (ED3) and IPTG were induced to express, and the recombinant protein was purified by 14 KD dialysis membrane gradient dialysis. Protein structural homology modeling was performed using the expasy protein online database. Signal P sever was used to predict the signal peptide sequence of ts IL-6 The gp130 binding site of tree shrew IL-6 was predicted by the structural fit of the template and the prediction model. Results The nucleotide and amino acid sequences of ts IL-6 shared higher homology with higher primates. The recombinant plasmid p ET-28a (+) - IL-6 was confirmed by double enzyme digestion and constructed correctly The relative molecular mass of the protein was about 24 000. The single objective band was purified at a concentration of 0.2 mg / ml. The spatial structure of hIL-6 and ts IL-6 proteins was similar. The gp130 binding site of ts IL-6Ⅱa Located between A and C helix, site Ⅲa is located in the flexible chain of A and B helices and near the C-terminal of the molecule. Conclusion The nucleotide and amino acid sequence of ts IL-6 is highly homologous with that of higher primates. The recombinant prokaryotic expression plasmid p ET-28a (+) - IL-6 was successfully constructed for subsequent IL-6-related pathology Research laid the foundation.