摘要： 为了深入研究己糖激酶（HK）的同工酶HK Ⅱ调控线粒体通透性转换孔（MPTP）的开放机理，通过RT-PCR技术和RACE技术，以克隆得到的肥城桃果实cDNA为模板，成功克隆得到HK Ⅱ基因全长，经原核表达和SDS-PAGE检测，体外构建原核表达载体pET-30a-HK Ⅱ ，成功地在大肠杆菌BL21（DE3）中进行表达；使用镍柱纯化法，得到纯化的重组蛋白。克隆得到HK Ⅱ基因共1 835 bp，其中编码区1 496 bp，共编码497个氨基酸。HK Ⅱ蛋白预测分子式为C₂₃₇₉H₃₈₄₆N₆₅₂O₇₁₇S₁₇，原子总数为7 611个，分子量为53.6 kDa，理论等电点为6.17，总平均疏水指数为0.023，表明HK Ⅱ蛋白为疏水性蛋白。TMHMM跨膜结构预测HK Ⅱ为跨膜蛋白，有1次跨膜，绝大部分在膜外。从二级结构预测结果来看，HK Ⅱ蛋白由15段α螺旋和11段β折叠和无规则卷曲构成。肥城桃HK Ⅱ蛋白预测的三级结构呈V字型，折叠结构较多。系统进化树和氨基酸相似性分析发现，桃果实HK Ⅱ与枇杷的相似度最高，达到了96.98%，桃果实HK Ⅱ与其他植物HK的氨基酸的同源性较高，说明HK Ⅱ具有较高的保守性。为进一步解析HK Ⅱ蛋白的结构和功能从而探讨HK Ⅱ调控线粒体MPTP的机理奠定了基础。

关键词: 肥城桃, 己糖激酶, 基因克隆, 生物信息学分析, 原核表达

Abstract: In order to study the regulation mechanism of hexokinase Ⅱ (HK Ⅱ) to mitochondrial permeability transition pore (MPTP) opening,a cDNA encoding HK Ⅱ protein was isolated from Feicheng peach fruits by RT-PCR and rapid amplification of cDNA ends (RACE).Induced by IPTG and determinated by SDS-PAGE,the expression vector pET-30a-HK Ⅱ was expressed by BL21.The HK Ⅱ protein was purified through nickel chelating affinity chromatography.The full-length cDNA of HK Ⅱ was 1 835 bp including 1 496 bp of coding sequence encoding a polypeptide of 497 amino acids.The predicted protein molecular formula of HK Ⅱ was C₂₃₇₉H₃₈₄₆N₆₅₂O₇₁₇S₁₇,containing 7 611 atoms.For HK Ⅱ,the molecular weight was 53.6 kDa,the theoretical isoelectric point was about 6.17,and the total average hydrophobicity index was 0.023,which indicated that HK Ⅱ protein was a hydrophobic protein.TMHMM transmembrane structure predicted that HK Ⅱ was a transmembrane protein with 1 transmembrane,most of which were outside the membrane.According to the results of the two level prediction,the HK Ⅱ protein was composed of 15 segments of α helix and 11 segments of β folding and the irregular curl.The three level structure of peach HK Ⅱ protein in Feicheng showed a V font,there were a large number of folded structure.It showed the highest homology with the loquat (96.98%) through the phylogenetic tree and multiple sequences alignment analysis.The high homology of amino acids of HK in peach fruit and other plants,indicating that HK Ⅱ is more conservative.The results would lay a foundation for further analysis of the structure and function of HK Ⅱ protein to explore the mechanism of HK Ⅱ regulation of mitochondrial MPTP.

Key words: Feicheng peach fruits, Hexokinase, Gene cloning, Bioinformatics analysis, Prokaryotic expression

中图分类号: 

• S662.03

• Q78