棉铃虫甾醇载体蛋白2原核表达及纯化

doi:10.3969/j.issn.1004-1524.2017.07.15

浙江农业学报 ›› 2017, Vol. 29 ›› Issue (7): 1166-1171.DOI: 10.3969/j.issn.1004-1524.2017.07.15

棉铃虫甾醇载体蛋白2原核表达及纯化

杜新凯, 任娟, 胡珺, 王常高, 林建国, 蔡俊, 杜馨^*

湖北工业大学发酵工程教育部重点实验室,工业发酵湖北省协同创新中心,工业微生物湖北省重点实验室,湖北武汉 430068

收稿日期:2017-02-22 出版日期:2017-07-20 发布日期:2017-07-24
通讯作者: 杜馨,E-mail:99023801@qq.com
作者简介:杜新凯(1991—),男,河南灵宝人,硕士研究生,研究方向为发酵过程优化。E-mail:duxinkai78@163.com
基金资助:
国家自然科学基金项目(31401807); 湖北工业大学高层次人才科研启动项目(BSQD13002); 湖北工业大学大学生创新创业项目(201510500064)

Prokaryotic expression and purification of sterol carrier protein-2 from Helicoverpa armigera

DU Xinkai, REN Juan, HU Jun, WANG Changgao, LIN Jianguo, CAI Jun, DU Xin^*

Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan 430068, China

Received:2017-02-22 Online:2017-07-20 Published:2017-07-24

摘要/Abstract

摘要： 甾醇载体蛋白2(SCP-2)在昆虫体内甾醇吸收及运输过程中有着重要的生理功能。试验构建重组表达质粒pET-22b/HaSCP2,转化至E. coli BL21 (DE3)进行诱导表达,并对IPTG浓度、诱导时间及温度进行优化,经Ni柱分离纯化后分析其与胆固醇结合活性。SDS-PAGE分析表明,重组蛋白相对分子质量约为16 ku,且在25 ℃,IPTG终浓度0.2 mmol·L^-1下诱导10 h为最佳诱导表达条件。经荧光探针8-苯胺-1-萘磺酸(1,8-ANS)竞争分析,胆固醇与HaSCP-2结合半数有效浓度(EC₅₀)为29.03 μmol·L^-1。该研究为深入研究棉铃虫SCP-2的功能以及后续HaSCP-2抑制剂的筛选研究奠定了基础。

关键词: 棉铃虫, 甾醇载体蛋白2, 原核表达, 原核纯化, 原核优化

Abstract: Sterol carrier protein 2 (SCP-2) plays important physiological roles in sterols absorption and transport in insects. In this study, the recombinant expression plasmid pET-22b/HaSCP2 was constructed and transformed into E. coli BL21 (DE3) for inducible expression. Induction conditions of the IPTG concentrations, the induction time and temperature were optimized. The recombinant HaSCP-2 was purified using Ni²⁺-affinity column, and the protein functions of HaSCP-2 was tested by competitive displacement assay. SDS-PAGE analysis suggested that the molecular weight of the recombinant protein was about 16 ku, and the optimal expression conditions were at the temperature of 25 ℃ and at the final concentration of 0.2 mmol·L^-1 IPTG for 10 h. Fluorescent probe 8-aniline-1-naphthalenesulfonic acid (1,8-ANS) was used for competitive analysis, and 1,8-ANS was replaced by cholesterol bound to HaSCP-2 with a half effective concentration (EC₅₀) of 29.03 μmol·L^-1. This study provides good opportunity for further study on the function of HaSCP-2 and the screening of HaSCP-2 specific inhibitors.

Key words: Helicoverpa armigera, sterol carrier protein-2, prokaryotic expression, prokaryotic purification, prokaryotic optimization

中图分类号:

S435

杜新凯, 任娟, 胡珺, 王常高, 林建国, 蔡俊, 杜馨. 棉铃虫甾醇载体蛋白2原核表达及纯化[J]. 浙江农业学报, 2017, 29(7): 1166-1171.

DU Xinkai, REN Juan, HU Jun, WANG Changgao, LIN Jianguo, CAI Jun, DU Xin. Prokaryotic expression and purification of sterol carrier protein-2 from Helicoverpa armigera[J]. , 2017, 29(7): 1166-1171.

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棉铃虫甾醇载体蛋白2原核表达及纯化

Prokaryotic expression and purification of sterol carrier protein-2 from Helicoverpa armigera

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