摘要
【目的】探究苦荞蔗糖合酶结构与功能的关系,构建表达载体以实现SuSy基因在大肠杆菌中的表达,并经亲和层析纯化后得到重组SuSy蛋白,为进一步研究该酶的结构和功能奠定基础.【方法】以苦荞(Fagopyrum tataricum)cDNA文库中克隆得到的蔗糖合酶基因重组质粒为模板,PCR扩增得到SuSy编码序列,将其与原核生物表达载体pET28a相连接构建了重组表达载体pET28a-SuSy,经EcoRⅠ和SalⅠ双酶切及测序鉴定正确后将其转入大肠杆菌(Escherichia coli)感受态细胞BL21,经异丙基-β-D-硫代半乳糖苷诱导表达SuSy重组蛋白,并对其诱导表达条件进行优化.【结果】在37℃、1.2 mmol/L IPTG诱导10 h时,SuSy蛋白表达量最高,分子量大小约为53.4 ku,超声破碎后经SDS-PAGE检测到该蛋白以包涵体形式存在.【结论】利用Co2+亲和柱层析纯化和His-tag鉴定得到了高纯度的重组SuSy蛋白,为该酶结构与功能的研究及多克隆抗体制备奠定了基础.
【Objective】To realize expression of SuSy gene in Escherichia coli by constructed expression vector,and purify its recombinant protein by affinity chromatography in order to study the relationship between structure and function of synthase sucrose of buckwheat.【Method】Sucrose synthase(SuSy)gene was amplified from cDNA library of buckwheat by PCR and fused into the prokaryotic expression vector pET28a.After screening and sequencing,the recombinant vector pET28a-SuSy was transformed into E.coli BL21 and expressed by the induction of IPTG.【Result】SDS-PAGE showed that the recombinant protein were more highly expressed at 37℃for 10 h with 1.2 mmol/L IPTG.It existed as incusion bodies after ultrasound treated,and the molecular weight was 53.4 ku.【Conclusion】The recombinant protein was successfully purified with Co2+affinity chromatography and His-tag identification.The research could lay a foundation for study structure and function of synthase sucrose and the preparation polyclnal antibodies.
作者
李慧婧
魏玉梅
吴慧昊
LI Hui-jing;WEI Yu-mei;WU Hui-hao(Experimental Teaching Department,Northwest Minzu Uiniversity,Lanzhou 730000,China)
出处
《甘肃农业大学学报》
CAS
CSCD
北大核心
2020年第3期71-77,共7页
Journal of Gansu Agricultural University
基金
中央高校基本科研业务费专项(31920160056)。
关键词
苦荞
蔗糖合酶
原核表达载体
纯化
Fagopyrum tatarium
sucrose synthase
prokaryotic expression vector
purification
