In order to construct and express human cardiac troponin C-linker-troponin I(P) fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pColdⅠ-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni2+ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo FinecareTM cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37℃and 4 or more months at 25℃and 4℃. 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.
Citation：
SONGXiaoli, LIUXiaoyun, CAILei, WUJianwei, WANGJihua. Construction of cTnC-linker-TnI(P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein. Journal of Biomedical Engineering, 2015, 32(6): 1267-1272. doi: 10.7507/1001-5515.20150225
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于志江, 陈旭, 欧阳藩.冷冻干燥技术在基因工程药物中的应用[J].生物加工过程, 2005, 3(2):58-63.
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- 1. 王建英, 贺洪军.心肌肌钙蛋白I(cTnI)在诊断急性心肌梗塞中的应用及意义[J].医学信息, 2010, 23(10):11-12.
- 2. DOI N, YANAGAWA H. STABLE:protein-DNA fusion system for screening of combinatorial protein libraries in vitro[J]. FEBS Lett, 1999, 457(2):227-230.
- 3. FITZGERALD K. In vitro display technologies-new tools for drug discovery[J]. Drug Discov Today, 2000, 5(6):253-258.
- 4. 尹焕才, 唐玉国, 王弼陡, 等.心肌肌钙蛋白I与AMI诊断及其检测方法研究综述[J].现代生物医学进展, 2010, 10(19):3782-3785.
- 5. CHRISTENSON R H, DUH S H, APPLE F S, et al. Standardization of cardiac troponin I assays:round robin of ten candidate reference materials[J]. Clin Chem, 2001, 47(3):431-437.
- 6. KATRUKHA A G, BEREZNIKOVA A V, FILATOV V L, et al. Degradation of cardiac troponin I:implication for reliable immunodetection[J]. Clin Chem, 1998, 44(12):2433-2440.
- 7. 刘大彪, 张明明, 卢仁泉, 等.重组人心肌TnI-TnC复合物的特性及其初步应用[J].放射免疫学杂志, 2008, 21(1):83-86.
- 8. 才蕾, 武建伟, 邓秋菊, 等.人心肌肌钙蛋白I冻干质控品的制备及评价[J].临床检验杂志, 2014, 32(8):615-617.
- 9. TATE J R, HEATHCOTE D, KOERBIN G, et al. The harmonization of cardiac troponin I measurement is independent of sample time collection but is dependent on the source of calibrator[J]. Clin Chim Acta, 2002, 324(1-2):13-23.
- 10. 于志江, 陈旭, 欧阳藩.冷冻干燥技术在基因工程药物中的应用[J].生物加工过程, 2005, 3(2):58-63.
