人心型脂肪酸结合蛋白(H-FABP)的原核表达、纯化和冻干品的制备

doi:10.13523/j.cb.20140311

中国生物工程杂志

2014, Vol. 34

Issue (3): 79-83 DOI: 10.13523/j.cb.20140311

技术与方法

人心型脂肪酸结合蛋白(H-FABP)的原核表达、纯化和冻干品的制备

武建伟, 才蕾, 任艳娜, 钱伟, 王继华, 唐时幸

广州万孚生物技术股份有限公司广州 510663

The Expression and Purification of Human Heart-type Fatty Acid Binding Protein and Preparation of Lyophilized Protein

WU Jian-wei, CAI Lei, REN Yan-na, QIAN Wei, WANG Ji-hua, TANG Shi-xing

Guangzhou Wondfo Biotech Co.Ltd., Guangzhou 510663, China

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摘要：

目的：获得重组人心型脂肪酸结合蛋白，分析其活性及制备冻干品。方法：从GenBank中检索人源H-FABP CDs 序列，合成基因后构建原核表达载体pET-28a（+）-H-FABP。将表达载体转入E.coli BL21（DE3），摸索pET-28a（+）-H-FABP最佳诱导条件，表达并纯化重组蛋白。使用检测试剂检测纯化后的重组H-FABP活性，研究最佳冻干方案并考察冻干品的稳定性。结果：经过优化诱导条件，重组H-FABP在BL21（DE3）中以可溶性蛋白形式表达。诱导条件为OD₆₀₀≈0.6，IPTG终浓度0.4mmol/L，30℃诱导4h。通过Ni²⁺亲和层析纯化可得到纯度大于95%的重组H-FABP蛋白。重组H-FABP冻干品可以在37℃稳定保存12d，25℃、4℃稳定保存至少4个月。结论：本项研究中的重组H-FABP表达体系成熟、蛋白活性高，冻干品稳定性好，为后续研究提供了稳定高效的生物原材料。

关键词： 心型脂肪酸结合蛋白; 原核表达; 冻干品; 急性心肌梗死

Abstract:

Objective: To obtain recombinant Human Heart-type Fatty Acid Binding Protein, detect its activity and prepare lyophilized protein. Methods: The CDs of Human H-FABP was searched from GenBank, synthesized into cloning vector. pET-28a(+)-H-FABP was constructed and transformed into E.coli BL21(DE3). After the optimal induction condition being researched, plenty of recombinant H-FABP was induced. Recombinant H-FABP was purified by Ni Sepharose 6 Fast Flow affinity chromatography and prepared into lyophilized protein. The activity of purified recombinant H-FABP and its lyophilized protein were detected by Wondfo H-FABP Test. Results: At the optimized induction condition(OD₆₀₀/0.6, IPTG/0.4mmol/L, Induced for 4h at 30℃), recombinant H-FABP was expressed as soluble protein in E.coli BL21(DE3). High activity recombinant H-FABP was obtained with over 95% purity by Ni-chelating affinity chromatography. Lyophilized protein of recombinant H-FABP was stable for 12 days at 37℃ and 4 or more months at 25℃. Conclusion: The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

Key words: Heart fatty acid binding protein Prokaryotic expression Lyophilized protein Acute myocardial infarction

收稿日期: 2013-12-24 出版日期: 2014-03-25

ZTFLH:

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通讯作者: 唐时幸 E-mail: tang.shixing@wondfo.com.cn

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引用本文:

武建伟, 才蕾, 任艳娜, 钱伟, 王继华, 唐时幸. 人心型脂肪酸结合蛋白(H-FABP)的原核表达、纯化和冻干品的制备[J]. 中国生物工程杂志, 2014, 34(3): 79-83.

WU Jian-wei, CAI Lei, REN Yan-na, QIAN Wei, WANG Ji-hua, TANG Shi-xing. The Expression and Purification of Human Heart-type Fatty Acid Binding Protein and Preparation of Lyophilized Protein. China Biotechnology, 2014, 34(3): 79-83.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140311 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I3/79

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