摘要
优化重组人β-catenin(138~686 aa)在大肠埃希菌中的原核表达条件,经分离纯化后进行生物学活性鉴定。利用基因工程技术,将构建的重组质粒β-catenin-pET-30a(+)转化到Escherichia coli Rosetta(DE3)中,经IPTG诱导后进行β-catenin原核表达。为了提高β-catenin表达量,从诱导时间、诱导温度与IPTG诱导浓度3个单因素方面进行原核表达条件优化。β-catenin经HisTrap层析柱分离纯化后,以荧光偏振实验和酶联免疫吸附测定实验(enzyme-linked immunosorbent assay,ELISA)进行生物学活性鉴定。构建的工程菌经原核表达条件优化后,确定诱导温度25℃、诱导时间10 h、0.2 mmol/L IPTG作为β-catenin最佳原核表达条件。荧光偏振实验和ELISA实验说明,纯化的β-catenin具有良好的生物学活性。本研究成功进行了重组人β-catenin原核表达条件的优化与生物学活性鉴定,为深入研究β-catenin在肿瘤免疫抵抗中的生物学功能奠定了实验基础。
Prokaryotic expression condition of recombinant humanβ-catenin(138-686 aa)in E.coli was optimized,and carried out biological characterization after isolation and purification.The constructed recombinantβ-catenin-pET-30a(+)plasmid was transformed into E.coli Rosetta(DE3)adopting genetic engineering technology and carried out prokaryotic expression ofβ-catenin after adopting IPTG induction.In order to enhance the expression amount ofβ-catenin,three single factors,induction time length,induction temperature,and IPTG induction concentration were carried out conditions optimization for prokaryotic expression.β-catenin was carried out characterization for biological activity after purification adopting HisTrap chromatographic column and ELISA determination.After the expression conditions optimization of the constructed prokaryotic engineered strain,the induction temperature was confirmed at 25℃,induction time at 10 h,and IPTG concentration at 0.2 mmol/L as the optimal conditions for prokaryotic expression.The experiment results of fluorescence polarization(FP)and ELISA showed that the purified-catenin had fine bioactivity.This study successfully carried out the prokaryotic expression conditions optimization of the recombinant humanβ-catenin and characterized its bioactivity,and had laid the experimental foundation for further research onβ-catenin in biological function for tumor immunity resistance.
作者
牛夏忆
韩茂椿
李淼
陈云雨
刘晓平
NIU Xia-yi;HAN Mao-chun;LI Miao;CHEN Yun-yu;LIU Xiao-ping(Inst.for Pharm.Screening&Evaluation,Wannan Med.Coll.,Wuhu 241002)
出处
《微生物学杂志》
CAS
CSCD
2020年第1期58-66,共9页
Journal of Microbiology
基金
国家自然科学基金项目(81703546)
安徽省自然科学基金项目(1808085QH265)
安徽省高校自然科学研究重大项目(KJ2019ZD30)
吉林省科技发展计划项目(20160520045JH)
皖南医学院博士科研启动基金项目(RCQD201617)
皖南医学院大学生科研资助金项目(WK2018S54)。
