[1]罗玄梅a,黄　薇a,孙高远b,等.应用基因密码子优化技术建立高效人降钙素原的原核表达方法及产品纯化和鉴定[J].现代检验医学杂志,2022,37(03):149-151.[doi:10.3969/j.issn.1671-7414.2022.03.031]
　LUO Xuan-meia,HUANG Weia,SUN Gao-yuanb,et al.High-efficiency Expression of Human Procalcitonin in Escherichia coli Based on Codon Optimization Technology and Purification and Identification of the Expressed Product[J].Journal of Modern Laboratory Medicine,2022,37(03):149-151.[doi:10.3969/j.issn.1671-7414.2022.03.031]
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应用基因密码子优化技术建立高效人降钙素原的原核表达方法及产品纯化和鉴定()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年03期
页码:
149-151
栏目:
研究简报·实验技术
出版日期:
2022-05-15

文章信息/Info

Title:
High-efficiency Expression of Human Procalcitonin in Escherichia coli Based on Codon Optimization Technology and Purification and Identification of the Expressed Product
文章编号:
1671-7414（2022）03-149-04
作者:
罗玄梅a; 黄　薇a; 孙高远b; 汤小琨b; 王璐瑶b; 邹丽辉a
（北京医院 a. 国家老年医学中心; b. 临床生物样本管理中心，北京 100730）
Author(s):
LUO Xuan-meia;  HUANG Weia;  SUN Gao-yuanb;  TANG Xiao-kunb;  WANG Lu-yaob;  ZOU Li-huia
(a. National Center of Gerontology; b. Clinical Biobank, Beijing Hospital, Beijing 100730, China)
关键词:
降钙素原; 参考物质; 重组蛋白质类
分类号:
Q503
DOI:
10.3969/j.issn.1671-7414.2022.03.031
文献标志码:
A
摘要:
目的　建立人降钙素原（ procalcitonin, PCT）表达体系，高效表达可溶性 PCT蛋白。方法　合成 PCT蛋白密码子优化序列，克隆至带麦芽糖结合蛋白（ maltose-binding protein, MBP）标签的 pRSF-Duet载体以构建 pMBP-PCT-pRSF原核表达质粒。在 E. coli BL21(DE3) pLysS菌株中诱导表达，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（ sodium dodecyl sulfate polyacrylamide gelelectrophoresis, SDS-PAGE）鉴定蛋白分子量及纯度，生物梅里埃 VIDAS全自动荧光免疫分析仪检测蛋白浓度、稳定性及均匀性。结果　pMBP-PCT-pRSF质粒成功构建及表达，浓度达 168（168.00±2.65）mg/L，纯度达 95%。在 4 ℃及 -20 ℃储存，PCT蛋白具有良好的稳定性（F=2.016，2.620，均 P＞ 0.05）和均匀性（F=0.727 3，0.973 9，均 P＞ 0.05）。结论　利用密码子优化，成功构建可溶性 PCT蛋白的高效原核表达体系，可充分满足临床实验室参考物质的需求。
Abstract:
Objective　To establish a prokaryotic expression system for human procalcitonin (PCT), and efficiently express and purify soluble PCT protein. Methods　The optimized PCT gene was synthesized and cloned into pRSF-Duet vector reside with maltose-binding protein (MBP) to generate pMBP-PCT-pRSF prokaryotic expression vector. Recombinant PCT was expressed in E. coli BL21 (DE3) pLysS. The molecular weight and purity of the protein were identified by SDS-PAGE, and the concentration and stability were analyzed by BioMerieux VIDAS automatic fluorescence immunoassay analyzer. Results　The vector pMBP-PCT-pRSF was successfully constructed. When expressed in E. coli BL21 (DE3) pLysS, the concentration of soluble PCT protein reached 168 (168.00±2.65) mg/L, and the purity reached 95%. Besides, recombinant PCT protein showed good stability (F=2.016, 2.620, all P ＞ 0.05) and homogeneity (F=0.727 3, 0.973 9, all P ＞ 0.05) when stored at 4 ℃ and –20℃ . Conclusion　By using codon optimization technology, the recombinant PCT protein was highly expressed in prokaryotes, which can fully meet the needs of clinical laboratory reference materials.

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备注/Memo

备注/Memo:
基金项目：国家自然科学基金（项目编号81871107）。
作者简介：罗玄梅（1997-），女，硕士在读，研究方向：老年退行性疾病的血管损伤修复与重构，E-mail:a1851590995@163.com。
通讯作者：邹丽辉（1982-），女，博士，E-mail:zouhui4371@hmoh.cn。
更新日期/Last Update: 1900-01-01