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

作　　者：冯凯[1] 李建平[1] 胡云光[1] 王晶晶[1] 王丽春[1] 李剑兰[1] 李琦涵[1] 刘龙丁[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《中国生物制品学杂志》2015年第6期557-563,共7页Chinese Journal of Biologicals

基　　金：云南省应用基础研究面上项目(2011FZ209);国家自然科学基金面上项目(81373142);北京协和医学院研究生创新基金项目(2013-1001-12)

摘　　要：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.

关 键 词：树鼩 白细胞介素-6 原核细胞 基因表达 同源建模 

分 类 号：R392.11[医药卫生—免疫学] Q786[医药卫生—基础医学]