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

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

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HFABP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。

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 BI21 （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 BI21 （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.