刺五加内生青霉鲨烯合酶蛋白表达和定位

Expression and Location Analysis of Penicillium minioluteum Squalene Synthase from Eleutherococcus senticosus

目的:对刺五加的内生菌青霉属Penicillium minioluteum P116-1a的鲨烯合酶(Squalene synthase,PmSS)进行原核表达和荧光定位。方法:将PmSS基因连接pET-30a(+),转化BL21(DE3)后诱导表达,通过SDS-PAGE分析表达效果。利用荧光表达载体pDsRed2-N1对PmSS蛋白定位。结果:成功构建了PmSS基因的体外表达载体pET-30a-PmSS,在BL21(DE3)能表达出约60 kDa的蛋白。温度为30℃,IPTG浓度为0.6 mmol/L时的表达量最高。荧光定位结果显示,PmSS呈点状或面状集中分布于内质网上。结论:获得了PmSS重组蛋白并确定了PmSS的表达部位,为进一步研究P.minioluteum提高刺五加皂苷含量的机制奠定基础。

Objective：To analysis the prokaryotic expression and fluorescence localization of squalene synthase （PmSS） isolated from Peni- cillium minioluteum. Method： PmSS gene was cloned into pET- 30a （ ＋ ）. The resultant expression vector was transformed into BL21 （ DE3 ）, then the induced expression product was analyzed by SDS - PAGE. And the location of PmSS protein in P. minioluteum was made sure by fluorescence expression vectors pDsRed2 -N1. Result：SS derived from P. minioluteum was expressed successfully. PmSS was ex- pressed in BL21 （ DE3 ）. A protein around 60 kDa was isolated. PmSS expressed better in 30℃ with IPTG concentration of 0. 6 mmol/ L Fusion expression product of PmSS and fluorescent protein in P. minioluteum was distributed as punctiform or facet. Condusion：PmSS protein was isolated and its location was detected. The result laid foundation for further study on the improvement mechanism of elenthero- side induced by P. minioluteum.