重组肌红蛋白的原核表达、纯化及其冻干质控品的制备

摘要/Abstract

摘要：

旨在为市场提供一种廉价、稳定、特异性强、灵敏度高的肌红蛋白质控品；将密码子优化后的肌红蛋白序列通过两步法进行化学合成，并连接到pET-28a表达载体上，转入大肠杆菌BL21（DE3），用0.5 mmol/L IPTG 37℃诱导表达；目的蛋白经Ni Sepharose 6 Fast Flow（FF）亲和层析进行纯化后，对其特异性进行检测，并研究蛋白稀释液的配方和优化冻干工艺，最后对冻干质控品的稳定性进行检测；结果显示目的蛋白分子量大小为20 kD左右，具有较强的特异性。蛋白冻干后具有很好的形态，37℃可以稳定保存10 d；25℃、4℃可以稳定保存4个月以上。该蛋白的冻干制品成本低、稳定性好、特异性强、灵敏度高，可用于定性定量肌红蛋白检测试剂盒的质控品。

关键词: 重组肌红蛋白, 原核表达, 纯化, 冻干, 质控品

Abstract:

We intended to provide a cheap, stable, highly specific and sensitive lyophilized protein as quality control for diagnositic kits. The myoglobin gene code was optimized and two-step methods were used in this study for the synthesis of myoglobin gene. The DNA fragment of myoglobin was inserted into pET-28a vector forming a recombination plasmid, then, transformed into BL21（DE3）bacteria. Protein expression was induced by 0.5 mmol/L IPTG and purified by Ni Sepharose 6 Fast Flow（FF）. After confirming its specificity, we studied the stability of the lyophilized protein at different storage temperatures（37℃, 25℃, 4℃）. Results showed that the target protein is approximately 20 kD with high specificity, good morphology and stability after freeze-drying under the optimized conditions and protein diluting solution. The lyophilized myoglobin protein with low cost, good stability, high specificity and sensitivity, canbe used as quality control for both qualitative and quantitative myoglobin testing kits.

Key words: Recombinant Myoglobin, Prokaryotic expression, Purification, Lyophilized, Quality control samples

任艳娜, 才蕾, 武建伟, 钱伟, 张荣华, 王继华, 唐时幸. 重组肌红蛋白的原核表达、纯化及其冻干质控品的制备[J]. 生物技术通报, 2014, 0(8): 189-195.

Ren Yanna, Cai Lei, Wu Jianwei, Qian Wei, Zhang Ronghua, Wang Jihua, Tang Shixing. The Expression and Purification of Recombinant Myoglobin and Preparation of Its Quality Control Samples[J]. Biotechnology Bulletin, 2014, 0(8): 189-195.

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