摘要
利用简并引物和RT-PCR方法从金针菇(Flammulina velutipes)幼嫩子实体中克隆获得FvGDH全长cDNA序列.构建入门载体pGWC-FvGDH,利用Gateway克隆技术的LR反应构建原核重组表达载体pDESTl7-FvGDH,转化大肠杆菌BL21(DE3).通过IPTG法诱导表达融合蛋白并进行表达条件优化.SDS-PAGE蛋白电泳分析表明,融合蛋白相对分子质量约为53 kD,与预测的一致.最佳表达条件为温度30℃、IPTG浓度0.4mmol/L、诱导4 h.融合蛋白表达量较高,实现了FvGDH的高效表达,并利用Western blotting对其特异性进行鉴定.FvGDH基因高效原核表达体系的成功建立,为进一步研究FvGDH的酶促动力学奠定了基础.
The full-length cDNA sequence of FvGDH gene was amplified from young Flammulina velutipes sporocarp by using a pair of degenerate primers and RT-PCR. Then the eDNA sequence was introduced into the vector pGWC for constructing entry vector pGWC-FvGDH. Finally, the prokaryotic expression vector pDEST17-FvGDH was obtained by the LR reaction of Gateway cloning system and transformed into E.coli BL21 （DE3）. The fusion protein expression was induced by IPTG, and the expression conditions were opti- mized. SDS-PAGE analysis indicated that the relative molecular mass of recombinant protein was about 53 kD as expected. The optimal condition for prokaryotic expression was performed at 30 ~C for 4 h with 0.4 mmol/L IPTG. The fusion protein displayed a high level among the whole bacterial proteins, which demon- strated that efficient expression of FvGDH was achieved. Protein specificity was also confirmed by Western blotting. In summary, an efficient prokaryotie expression system of FvGDH has been successfully established, which provides a foundation for further research on the enzyme kinetics of FvGDH.
出处
《生命科学研究》
CAS
CSCD
北大核心
2013年第3期230-237,共8页
Life Science Research
基金
国家转基因生物新品种培育重大专项项目(2009ZX08001-030B)
国家自然科学基金面上项目(31170172)
湖南省自然科学基金资助项目(12JJ3024)
植物分子遗传国家重点实验室开放课题
中央高校基本科研业务费湖南大学重大前期培育项目
青年教师科技创新扶持项目资助
