黄瓜脂氧合酶基因CsLOX2的原核表达载体的构建及表达分析

Construction and Expression of a Cucumber Lipoxygenase Gene CsLOX2 in Prokaryotic Cells

以先前克隆到的黄瓜脂氧合酶基因CsLOX2全长的cDNA为模板,用含有特异性酶切位点的Yh1、Yh2为引物,通过PCR方法将黄瓜脂氧合酶基因CsLOX2的ORF区构建到大肠杆菌表达载体pGEX-4T-1上获得原核表达载体p4t-LOX2,将该表达载体p4t-LOX2转化到大肠杆菌菌株BL21(DE3)中,获得相应的重组工程菌。在IPTG诱导下,通过SDS-PAGE电泳,得到一条约115kD的融合蛋白条带,除去pGEX-4T-1自身诱导的约26kD大小的GST标签蛋白后,CsLOX2编码一个约89kD的蛋白。经过融合蛋白表达体系的优化分析,表明该融合蛋白在37℃,0.80mmol·L-1IPTG诱导表达10.5h,可获得融合蛋白的最大表达量。

The ORF of cucumber(Cucumis sativus L.)CsLOX2 gene was inserted into an expression vector pGEX-4T-1 through PCR method using specific primers Yh1 and Yh2,and the recombined expression vector p4t-LOX2 was constructed. The expression amount of fusion protein was detected under different expressive conditions,such as different time,different temperature and different concentration of IPTG. The 115 kD fusion protein was obtained and induced by IPTG and it showed that CsLOX2 gene encoded a 89 kD protein after the 26 kD GST protein tag was removed.The proper induction condition was 37 ℃ temperature,0.80 mmol·L -1 IPTG concentration and 10.5 h inducing time.