摘要
目的以生物信息学预测细粒棘球绦虫甘油醛3-磷酸脱氢酶(EgGAPDH)编码基因及其编码蛋白的结构和功能,利用大肠杆菌原核表达系统高效表达EgGAPDH重组蛋白,体外检测其酶活性。方法运用各软件对EgGAPDH的理化性质、保守功能域、系统发生树和二级结构、三级结构等方面进行分析后,将EgGAPDH基因亚克隆于表达载体pET30a(+),转化入E.coli BL21(DE3)感受态细菌,表达、纯化重组蛋白,并利用底物3-磷酸甘油醛测定重组蛋白EgGAPDH的酶催化活性。结果 EgGAPDH cDNA序列长度为1 011bp,编码一个336个氨基酸的蛋白。该GAPDH蛋白理论相对分子质量为36 128.3Da,等电点为8.44,具有GAPDH蛋白家族的两个功能结构域,富含6种类型的特定功能位点。成功构建的原核表达载体pET30a(+)-EgGAPDH在E.coli BL21(DE3)中高效表达,且发现表达产物主要以可溶形式分布于裂解上清,制备甘油醛3-磷酸脱氢酶纯品并通过酶活检测显示其具有高效酶活性。结论 EgGAPDH基因可在原核系统中获得有酶催化活性的高效表达,为进一步研究其在糖代谢中的功能奠定了基础。
The gene encoding Echinococcus granulosus glyceraldehyde 3-phosphate dehydrogenase （EgGAPDH） and the structure and function of the encoded protein using bioinformatics were predicted in this study. And the EgGAPDH protein in E. coli prokaryotie expression system was cloned and expressed and assessed the enzyme activity of the protein in vitro. Ac- cording to the predicted results such as physical and chemical properties, conserved domains, phylogenetic tree, and the sec- ondary structure and 3D structure, the EgGAPDH gene was subeloned into high-level expressed vector pET30a（＋）, and the recombinant plasmid was subsequently transformed into competent E. coli BL21 （DE3）. The recombinant protein was expressed and purified. And its catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate （3- GAP）. Results showed that the EgGAPDH eDNA, encoding a 336 amino acid protein with a predicted molecular weight of 36 128.3 Da and PI value of 8.44, was composed of 1 011 bp. The EgGAPDH protein was composed of 6 kinds of functional sites and 2 typical GAPDH family functional domains. The recombinant plasmid pET30a（＋）-EgGAPDH was constructed and the EgGAPDH protein was effectively expressed in supernatant of the lysis. The enzyme activity assay revealed that the puri- fied EgGAPDH had a significant enzyme activity in vitro. In conclusion, the EgGAPDH gene can express in prokaryotic ex- pression system efficiently with definite enzyme activity and it provides a significant reference for further exploration of its func- tion in glucose metabolism.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2014年第2期125-129,共5页
Chinese Journal of Zoonoses
基金
国家自然基金资助项目(No.81171632)资助~~
关键词
细粒棘球绦虫
甘油醛3-磷酸脱氢酶
生物信息学
原核表达
酶活性检测
Echinococcus granulosus glyceraldehyde-3-phosphate dehydrogenase （GAPDH） bioinformatics prokary-otic expression enzyme activity
