目的 构建芍药Paeonia lactiflora Actin(PlActin)基因原核表达载体, 优化PlActin基因在大肠杆菌中的高效表达体系。方法 基于PlActin基因cDNA序列及原核表达载体pET-32a (+) 多克隆位点序列, 设计一对5'末端分别插入BamH I和Hind III酶切位点的特异性PCR引物, 基于RT-PCR技术亚克隆PlActin基因;用BamH I和Hind III双酶切重组质粒pMD18-T-PlActin和原核表达载体pET-32a (+), 胶回收纯化目的基因和空载体, 连接、转化后应用菌落PCR、质粒PCR和双酶切鉴定出重组子pET-32a (+)-PlActin, 转化BL21(DE3)菌株获得基因表达工程菌;分别设置诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)浓度梯度、诱导的菌体起始密度梯度及表达时间梯度, 诱导PlActin基因在大肠杆菌中表达, SDS-PAGE电泳、考马斯亮蓝R250染色后制备干胶并比较重组Actin蛋白的表达量。结果 亚克隆测序结果表明PlActin与目的序列完全一致, 且其编码区序列(CDS)上下游成功插入了BamH I和Hind III酶切位点;成功构建了芍药原核表达载体pET-32a (+)-PlActin;诱导剂IPTG的最佳浓度为0.1 mmol/L, 诱导的菌体起始密度(A₆₀₀)为0.4～0.6, 最佳表达时间为4 h。结论 构建了PlActin基因原核表达载体pET-32a (+)-PlActin, 建立了PlActin基因在大肠杆菌中的高效表达体系。

Objective To construct the prokaryotic expression vector of Actin gene and to establish the high-level expression of Actin of Paeonia lactiflora in Escherichia coli. Methods Based on cDNA sequence of Actin of P. lactiflora and the polyclonal sites on the prokaryotic expression vector pET-32a (+), a pair of PCR primers whose 5' end was inserted with BamH I and Hind III, respectively were designed. Subcloning of Actin was carried out using RT-PCR technique. The recombinant plasmid pMD18-T-PlActin and the prokaryotic expression vector pET-32a (+) was digested by BamH I and Hind III and the objective gene and the empty vector were purified. After ligation and transformation, the recombinant pET-32a (+)-PlActin, which was characterized by colony PCR, plasmid PCR, and double enzyme digestion, was transformed into BL21 (DE3), and the engineering expression strain was obtained. The expression of recombinant Actin protein in E. coli was induced at different concentration of inducer IPTG, different bacteria density, and different expression time. After SDS-PAGE and Coomassie brilliant blue R250 staining, the dry gel was prepared and the expression level of recombinant Actin protein was analyzed. Results Subcloning sequencing result showed that the sequence of PlActin was exactly same with the objective sequence and the 5' and 3' ends were successfully inserted with BamH I and Hind III sites. The prokaryotic expression vector of Actin gene pET-32a (+)-PlActin was constructed successfully. The best concentration of IPTG was 0.1 mmol/L and the bacteria density A₆₀₀ was 0.4 to 0.6. The optimal expression time was 4 h. Conclusion The prokaryotic expression vector of Actin gene pET-32a (+)-PlActin is constructed and the high-level expression of Actin of P. lactiflora in E. coli is established.

国家自然科学基金资助项目(U1204323);河南省科技厅国际合作项目(134300510052)