白念珠菌酰基载体蛋白1的制备

doi:10.16150/j.1671-2870.2018.06.005

诊断学理论与实践 ›› 2018, Vol. 17 ›› Issue (06): 645-649.doi: 10.16150/j.1671-2870.2018.06.005

白念珠菌酰基载体蛋白1的制备

孟玲宁^1,2, 刘锦燕², 王钰婷^1,2, 项明洁^1,2

1.上海交通大学医学院附属瑞金医院检验科, 上海 200025;
2.上海交通大学医学院附属瑞金医院卢湾分院放免检验科, 上海 200020

收稿日期:2018-08-02 出版日期:2018-12-25 发布日期:2018-12-25
通讯作者: 项明洁 E-mail: mjxiang123456@126.com
基金资助:
上海市医学重点专科（ZK2012A21）; 上海市卫计委课题（201740069）; 上海市卫计委课题（2018 40227）; 上海市黄浦区卫计委课题（HKM201702）

Preparation of recombinant protein acyl carrier protein 1(Acp1) of Candida albicans

MENG Lingning^1,2, LIU Jinyan², WANG Yuting^1,2, XIANG Mingjie^1,2

1. Department of Clinical Laboratory, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;
2. Radioimmunology and Clinical Laboratory, Luwan Branch, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200020, China

Received:2018-08-02 Online:2018-12-25 Published:2018-12-25

摘要/Abstract

摘要： 目的：通过在大肠埃希菌BL21中表达白念珠菌酰基载体蛋白1（acyl carrier protein 1, Acp1）, 以制备高纯度的目的：蛋白。方法：采用PCR扩增目的：基因ACP1后, 在序列的C端拼接上6xHis tag及终止子, 拼接完成后连接构建到质粒中, 成为原核表达载体pET30a-ACP1, 转化入大肠埃希菌感受态细胞BL21中, 通过异丙基-β-D-硫代半乳糖苷（isopropyl-beta-D-thiogalactopyranoside, IPTG）诱导表达。收集表达产物后通过镍离子螯合柱纯化, 最终利用考马斯亮蓝Bradford法测得目的：蛋白含量。结果：白念珠菌酰基载体蛋白Acp1在大肠埃希菌BL21中高效表达;抽提纯化蛋白后行蛋白电泳检测, 结果显示, 在还原条件下Acp1一直存在2条相对分子质量为17 000的条带。经还原和非还原电泳及蛋白印迹法检测, 结果显示该蛋白在非还原状态下有聚集。利用Bradford方法测得蛋白Acp1浓度为2.68 mg/mL。结论：成功制备了白念珠菌酰基载体蛋白Acp1, 为后期利用荧光偏振法筛选靶点为Ppt2酶的临床药物提供基础。

关键词: 白念珠菌, 酰基载体蛋白Acp1, 蛋白制备纯化

Abstract: Objective: To prepare a high purity target protein by expressing Candida albicans acyl carrier protein Acp1 in E. coli BL21. Methods: The target gene Acp1 was amplified by PCR, and then the 6XHis tag and terminator were ligated into the C-terminus of the sequence. After splicing, the sequence was inserted into the plasmid to form the prokaryotic expression vector pET30a-ACP1, which was then transferred into E. coli BL21. IPTG was added to induce the expression. The expression product was collected and purified by the column Ni, and finally the target protein content was measured by the Bradford method. Results: The Candida albicans acyl carrier protein Acp1 was highly expressed in E. coli BL21. The electrophoresis results showed that there were two bands of Acp1 under reducing conditions. After reduction and non-reduction electrophoresis and Western blotting, the results showed that the protein was aggregated in a non-reduced state. The protein Acp1 concentration was determined to be 2.68 mg/mL by the Bradford method. Conclusions: The Candida albicans acyl carrier protein Acp1 is successfully prepared, which provides a basis for the later screening of clinical drugs targeting Ppt2 by fluorescence polarization.

Key words: Candida albicans, Acyl carrier protein Acp1, Protein preparation and purification

中图分类号:

R446.5

孟玲宁, 刘锦燕, 王钰婷, 项明洁. 白念珠菌酰基载体蛋白1的制备[J]. 诊断学理论与实践, 2018, 17(06): 645-649.

MENG Lingning, LIU Jinyan, WANG Yuting, XIANG Mingjie. Preparation of recombinant protein acyl carrier protein 1(Acp1) of Candida albicans[J]. Journal of Diagnostics Concepts & Practice, 2018, 17(06): 645-649.

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白念珠菌酰基载体蛋白1的制备

Preparation of recombinant protein acyl carrier protein 1(Acp1) of Candida albicans

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