树鼩白细胞介素-6的原核表达及其分子特征

Prokaryotic expression and molecular character of tree shrew IL-6

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 tree shrew IL-6（ts IL-6） in prokaryotic cells and analyze the molecular characters of expressed protein. Methods The common sequences of IL-6 m RNAs of human, monkey and rodents were blasted in the c DNA library of tree shrew. The obtained nucleotide sequence of ts IL-6 was translated to amino acid sequence, both of which were compared by MEGA 6 software, and a phylogenetic tree was plotted. The PCR primers of ts IL-6 was designed according to the predicted IL-6 gene sequence, and the PCR product was inserted into prokaryotic expression vector p ET-28-a（＋）. The constructed recombinant plasmid p ET-28a（＋）-IL-6 was transformed to competent E. coli BL21（DE3）and induced with IPTG. The expressed protein was purified by dialysis with 14 k D dialyzer. Homologous modeling of protein structure was performed by on-line data bank expasy, and the signal peptide sequence of ts IL-6 was predicted by Signal P sever. The gp130 binding site of ts IL-6 was predicted by the structural fitting of template and predicted model. Results The nucleotide and amino acid sequences of ts IL-6 were highly homologous to those of higher primates. Restriction analysis proved that recombinant plasmid p ET-28a（＋）-IL-6 was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 24 000, showed a single band on SDS-PAGE profile after purification, of which the concentration was 0. 2 mg / ml. The space structure of h IL-6 was similar to that of ts IL-6. The gp130 binding site Ⅱa was between the helixes A and C, while site Ⅲa at the flexible linking regions of helixes A and B as well as the region near to the C-terminus. Conclusion The nucleotide and amino acid sequences of ts IL-6 were highly homologous to those of higher primates. Recombinant plasmid p ET-28a（＋）-IL-6 was successfully constructed, which laid a foundation of further pathological study on IL-6.