杜仲EuCHIT1基因表达载体构建及在大肠杆菌中的表达

构建了T7启动子驱动杜仲几丁质酶基因EuCHIT1的原核表达载体pET-EuCHIT1和pMCSG-EuCHIT1,其表达产物分别含6个组氨酸(6His)标签和6个组氨酸连接的麦芽糖结合蛋白(his6-tag–maltose-binding protein,MBP)标签,分别将重组载体遗传转化大肠杆菌细胞BL21(DE3),在37℃、150 rpm条件下培养至菌液OD值为0.4~0.8后,转到16℃、150 rpm条件下以1 m MIPTG诱导培养12 h,表达产物经SDS-PAGE分析,p ET-EuCHIT1/BL21(DE3)表达以包涵体形式存在36.03 kD融合蛋白,pMCSG-EuCHIT1/BL21(DE3)成功表达可溶形式存在的77.21 kD融合蛋白。故可以使用pMCSG-EuCHIT1/BL21(DE3)获得可溶的融合蛋白,为之后的EuCHIT1的多克隆抗体的制备和及功能研究奠定基础。

Construction of Eucommia ulmoides EuCHIT1 gene expression vectors and its expression in Escherichia coli

The prokaryotic expression vectors pET-EuCHIT1 and pMCSG-EuCHIT1 of Eucommia ulmoides chitinase gene ( EuCHIT1) were constructed with strong T7 promoter. The expression products of pET-EuCHIT1 and pMCSG-Eu-CHIT1 contained six histidine (6His) tags and six maltose-binding proteins (his6-tag-maltose-binding protein, MBP), respectively. The recombinant vectors were transformed into E.coli cell strain BL21(DE3) for expression. The transformed cell were cultured under the conditions of 37℃ and 150 r until OD value of the bacteria was 0.4~0.8, then induced for 12 h by 1 mmol/L IPTG under the conditions of 16℃ and 150 r. The expression proteins were analysed by SDS-PAGE. The expression of pET-EuCHIT1/BL21(DE3) contained 36.03 kD fusion protein, and pMCSG-EuCHIT1/BL21(DE3) DE3) successfully expressed the soluble 77. 21 kD fusion protein. It is concluded that the chtinase gene ( EuCHIT1) could be expressed in pMCSG-EuCHIT1/BL21( DE3) to obtain soluble fusion protein, thus laying a foundation for prepa-ration of EuCHIT1 polyclonal antibody and for study of its function.