家蚕微孢子虫己糖激酶基因的克隆及序列结构分析和原核表达

Cloning, Sequence Structure Analysis and Prokaryotic Expression of Nosema bombycis Hexokinase Gene

己糖激酶（hexokinase,HXK）是糖酵解途径的限速酶,在能量代谢中充当极其重要的角色。家蚕微孢子虫（Nosema bombycis）具有完整的糖酵解途径,其基因组中有2个序列相似度较高的己糖激酶编码基因拷贝（NBO_1320g0001,NBO_27g0008）。根据其中的一个基因拷贝（NBO_27g0008）序列设计特异性引物,以家蚕微孢子虫基因组DNA为模板成功克隆了该基因,命名为Nb HXK-1。该基因大小为894 bp,包含完整的ORF,编码297个氨基酸,预测蛋白质的分子质量为34.241 k D,等电点（p I）为5.26,蛋白质的氨基酸序列有31个磷酸化位点和4个潜在的N-糖基化位点,无跨膜结构域,有信号肽,二级结构主要由α螺旋（54.55%）、延伸片段（17.80%）和无规则卷曲（27.61%）组成。系统进化分析显示Nb HXK-1与东方蜜蜂微孢子虫（Nosema ceranae）己糖激酶（Nc HXK）氨基酸序列的亲缘关系较近。将Nb HXK-1基因插入原核表达载体p ET-28a（＋）并转化BL21（DE3）感受态细胞,用IPTG诱导表达后经蛋白质串联质谱鉴定和Western blot分析显示,获得了与预期大小一致的约35 k D的重组Nb HXK-1蛋白,为进一步阐明Nb HXK-1的生物学功能奠定了基础。

Hexokinase （HXK） is a rate-limiting enzyme in glycoiysis pathway and plays an important role in energy me- tabolism. Nosema bombycis has a complete glycolytic pathway. HXK of Nosema bombycis has two gene copies （NBO_ 1320g0001 and NBO_27g0008） with high sequence similarity. Specific PCR primers for one of the gene copies （NBO_ 27g0008） were designed. The corresponding gene was successfully cloned and named as NbHXK-1. The ORF of Nb- HXK-1 gene is 894 bp long and encodes 297 amino acids, The predicted molecular weight and isoelectric point of Nb- HXK-1 are 34. 241 kD and 5.26, respectively. NbHXK-1 contains 31 phosphoryiation sites and 4 potential N-glycosylation sites. It has no transmembrane domain but has signal peptide. Its secondary structure mainly consists of a-helix （54.55%）, extended strand （17.80%） and random coil （27.61%）. Phvloqenetic analysis showed that NbHXK has aclose genetic relationship with Nosema ceranae Hexokin- ase. NbHXK-1 gene was cloned into pET-28a（＋） expres- sion vector and transformed into BL21 （DE3） for prokary- otic expression. After induction with IPTG, protein with the expected size of approximately 35 kD was obtained as characterized by tandem mass spectrometry and Western blot analysis. These data provide a good basis for furtherstudies on biological function of NbHXK-1.