根据基因组序列信息，利用cDNA末端快速扩增技术（rapid-amplification of cDNA ends，RACE）得到了红色红曲菌中组蛋白去乙酰化酶mrsir2基因的完整cDNA序列，其编码序列（coding sequence，CDS）为1539 bp，编码512个氨基酸，含有一个SIR2蛋白保守结构域。根据大肠杆菌的密码子的偏好性对mrsir2序列进行优化，优化后的序列与pET-28b载体连接后转入宿主菌E. coli BL21中进行IPTG诱导表达，并优化表达条件。实验结果表明：在16 ℃条件下用终浓度为0.25 mM的IPTG诱导培养16 h后，目的蛋白MrSir2可溶性表达效果良好。Ni2+柱亲和层析纯化后的MrSir2重组蛋白在SDS-PAGE上显示为一条约75 ku大小的条带，蛋白定量浓度达1.97 mg/mL，经Western blot鉴定为目的蛋白，测定酶活为78.5(OD/min/mg)，780 μM的二氢香豆素（dihydrocoumarin，DHC）对MrSir2蛋白的酶活抑制率为47%。MrSir2蛋白的可溶性表达为全面了解其酶学特征提供了材料，也为体外分析蛋白相互作用奠定了基础。

According to the sequenced genomic DNA information, the whole cDNA sequence of mrsir2 gene was obtained by rapid-amplification of cDNA ends (RACE) corresponding to coding sequence (CDS) with 1539 bp, which codes for 512 amino acids and contains a conserved domain of SIR2 protein. The CDS of mrsir2 was optimized according to the codon preference of E. coli. The CDS of mrsir2 was ligated to the pET-28b vector and then transferred into the host strain E. coli BL21 to be induced expression by IPTG. The expression condition was optimized. The experiment results showed that the target protein MrSir2 was expressed well in soluble way when the host was induced by IPTG at a final concentration of 0.25 mM for 16 h at 16 °C. The soluble recombinant protein was purified with Ni2+ column affinity chromatography. Result of SDS-PAGE showed a band with the molecular weight of approximate 75 ku.The protein concentration was as high as 1.97 mg/mL. The target protein was identified by western blot. The enzyme activity was 78.5 (OD/min/mg) detected by Epigenase? Universal SIRT Activity/Inhibition Assay Kit (Colormetric). The inhibition rate of 780μM dihydrocoumarin(DHC) to MrSir2 was 47%. The soluble expression of MrSir2 provided materials for understanding its enzymatic characteristics. Results will provide the foundation for exploring the interaction of protein-protein in vitro.

国家自然科学基金面上项目（31671835）；中央高校基本科研业务费专项资金资助项目（2662018PY090）