玉米(zea mays.L)ZmCen基因原核表达载体的构建及表达体系的优化

Construction Optimization of Expression Conditions of ZmCen Gene(zea mays L.) in Prokaryotic Cells

以玉米(zea mays L.)cDNA为模板,并将其构建到带有GST表达标签的原核表达载体pGEX-6p-1中,构建的重组载体pGEX-6p-Zmc e n转化至大肠杆菌BL21(DE3)。为表达出较大量的可溶性GST-Zm Cen融合蛋白,设置0.1 mmol/L,0.3 mmol/L和0.5 mmol/L IPTG3种不同浓度,诱导温度和4h、16h和20h的诱导时间建立正交体系,诱导产物经超声波裂解破碎细胞后,通过12%SDS-PAGE比较分析融合蛋白表达量,从而确定最佳的表达体系。结果显示:当诱导温度28℃,IPTG浓度为0.3mmol/L,诱导20 h时可以获得较大量的45kD可溶性融合蛋白(GST-Zm Cen)表达量。

The ORF of maize（zea mays L.）Zmcen gene was cloned into an expression vector p GEX-6p-1 through PCR method,and the GST recombined expression vector pG EX-6p- Zmcen was constructed. The recombinant expression conditions were optimized through orthogonal experiment under three different time,three different temperatures and three different concentrations of IPTG. The expression fusion protein was detected by 12 % SDS-PAGE. The 45 kD fusion protein was obtained and induced by IPTG. The optimization soluble induction condition was 28 ℃ temperature,0.3 mmol/L IPTG concentration and 20 h inducing time.