中文摘要：

目的：AMP激活的蛋白激酶（AMPK）在脑内作为多功能能量感受器发挥作用，主要协调代谢和能量的需要。脑神经元中AMPKα2亚基的含量明显高于α2亚基，本研究选择α2亚基作为对象，研究其在大肠杆菌中的表达情况，为以蛋白原核表达为前提的研究技术的应用提供实验依据。方法：大鼠AMPKet2蛋白编码区片段通过PCR方法从重组pcDNA3质粒中扩增后，克隆至带有λcI基因的pBT载体，构建融合表达载体pBT—AMPKα2。通过核苷酸序列测定证实结果正确后，转化pBT-AMPKα2入大肠杆菌XL—1 BlueMR菌株，IPTG诱导目的蛋白表达。结果：Western blotting显示AMPKα2-λcI融合蛋白表达，分子量约为89ku，与预期相符合。结果还表明AMPKα2-λcI融合蛋白以可溶性蛋白和不溶性包涵体两种形式存在，前者不稳定易发生降解，生成AMPKα2和kcI蛋白，分子量分别是62ku和27ku；后者稳定不发生降解。结论：AMPKα2亚基能以AMPKα2-λcI融合蛋白的形式在大肠杆菌中表达，这为今后以pBT—AMPKα2融合表达载体为工具利用大肠杆菌双杂交系统筛选AMPKα2的相互作用蛋白奠定基础。

英文摘要：

Objective： AMP-activated protein kinase （AMPK） plays a role in brain as a multifunctional energy sensor that regulates cellular metabolism and energy demand. Considering that the α2 catalytic subunit is more highly expressed than α1 catalytic subunit in neurons, this study chose α2 catalytic subunit to investigate its expression in Escherichia coli, which would provide the basis of experiments for further application of techniques based on the prokaryotic expression. Methods： A fragment encoding rat AMPKα2 was amplified by PCR from the recombinant pcDNA3 plasmid and fused in flame with the λcI of pBT vector to construct fusion expression vector pBT-AMPKα2. After confirmation with nucleotide sequence analysis, pBT-AMPKα2 vector was transformed into Escherichia coli XL-1 Blue MR and induced by IPTG for the expression of recombinant protein. Results： The AMPKα2-λcI fusion protein detected by Western blotting is approximately 89ku. Moreover, the fusion protein was expressed in two forms, soluble protein as well as inclusion body. The former was unstable and inclined to be broken down producing AMPKα2 and λcI proteins, approximate molecular weight is 62ku and 27ku, respectively. However, the latter was stable and not broken down. Conclusions： The study confirms expression of AMPKα2 subunit in the form of AMPKα2-λcI fusion protein in Escherichia coli, which may be a foundation for using fusion expression vector pBT-AMPKα2 as a tool and screening AMPKα2 interacting proteins by bacterial two-hybrid system.