家蚕精氨酸激酶原核表达纯化、结构与活性分析

Prokaryotic expression, purification and characterization of arginine kinase of Bombyx mori

摘要:

精氨酸激酶 (Arginine kinase，AK) 是无脊椎动物体内能量代谢的关键酶，在生长发育、营养利用、免疫抗性、胁迫应答等生命活动过程中发挥着重要的调控作用。家蚕精氨酸激酶BmAK与能量平衡、抗NPV病毒过程相关，但目前关于其分子结构和酶学性质的研究不多。克隆了BmAK基因ORF序列，分析了其染色体定位、基因组结构、mRNA结构、二级结构和三级结构。进化分析表明AK在进化过程中高度保守。原核表达获得了可溶性的BmAK重组蛋白，通过Ni-NTA亲和层析纯化了BmAK。圆二色光谱分析显示BmAK包含α螺旋结构，其α螺旋结构在pH 5–10范围内相对稳定。酶活分析表明BmAK的最适温度为30 ℃，最适pH为7.5。25 ℃时BmAK的催化活性最大，在15–30 ℃范围内，BmAK的结构相对稳定，活性差别不大。BmAK的结构在pH 7.0左右相对稳定。这些研究为揭示BmAK的结构和功能提供了基础，有助于开发以AK为分子靶标的绿色安全环保的新型杀虫剂。

Abstract:

Arginine kinase (AK) is a key enzyme in energy metabolism of invertebrates and plays an important regulatory role in the life activities such as growth and development, nutrition utilization, immune resistance and stress response. Arginine kinase of Bombyx mori (BmAK) is related to the energy balance and anti-NPV process, but there is little research on its molecular structure and enzymatic properties. We cloned the ORF sequence of BmAK gene, and analyzed chromosomal localization, genomic structure, mRNA structure, secondary and tertiary structure. Phylogenetic analysis indicated that AK was highly conserved in evolution. Soluble recombinant BmAK was obtained by prokaryotic expression, and purified by Ni-NTA affinity chromatography. The circular dichroism spectroscopy showed that BmAK contained α-helix structures, and its α-helix structures were relatively stable in the pH range between 5 and 10. Enzyme activity analysis showed that the optimum temperature of BmAK was 30 ℃ and the optimum pH of BmAK was 7.5. The optimal temperature of BmAK was 25 ℃. Between 15 ℃ and 30 ℃, the structure and activity of BmAK was relatively stable. The structure of BmAK was relatively stable at pH 7.0. Our findings reveal the structure and function of BmAK to develop novel green safe and environmentally friendly insecticides.