cTnC-linker-TnI(P)基因的构建、融合蛋白表达及冻干品的制备

Construction of cTnC-linker-TnI(P)Genes,Expression of Fusion Protein and Preparation of Lyophilized Protein

从GenBank中检索人源心肌肌钙蛋白C亚基(cTnC)和心肌肌钙蛋白I亚基(cTnI)编码序列,合成cTnC和cTnI第30至110氨基酸基因-cTnI(P),并在两者之间合成一段由15个氨基酸(G4S)3组成的接头(linker)序列。构建原核表达载体pColdⅠ-cTnC-linker-TnI(P),转化大肠杆菌BL21(DE3),用异丙基-β-D-硫代半乳糖苷(IPTG)诱导融合蛋白表达,结果在大肠杆菌中成功实现了cTnC-linker-TnI(P)融合蛋白的稳定可溶性高表达。经Ni 2+柱纯化后得到纯度大于95%的cTnC-linker-TnI(P)融合蛋白,制备冻干品并采用万孚飛测?心肌肌钙蛋白I(cTnI)定量检测试剂盒检测其稳定性。cTnC-linker-TnI(P)冻干品在37℃可稳定保存10d以上,25℃、4℃稳定保存4个月以上。本研究获得的cTnC-linker-TnI(P)融合蛋白活性高,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。

In order to construct and express human cardiac troponin C-linker-troponin I（P）[ cTnC-iinker-TnI（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, pCold I -cTnC-linker TnI（P） was constructed and transformed into E. coli BL21（DE3）. Then, cTnC-linker-TnI（P） fusion protein was induced by isopropyl-13-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 he provided as biological raw materials for further research.