斑马鱼TK1基因的克隆表达

Cloning and Expression of Thymidine Kinase from Zebrafish Danio rerio

目的:克隆斑马鱼TK1基因的cDNA序列,并在大肠杆菌中诱导表达,对其产物进行生物学活性鉴定。方法:采用RT-PCR和RACE方法,克隆TK1的cDNA全长序列。表达载体在大肠杆菌BL21(DE3)中进行诱导表达。表达蛋白利用镍离子柱纯化。结果:获得TK1基因的cDNA全长序列,编码一个分子量为26kD的蛋白。结论:TK1融合蛋白在28℃条件表现出比较高的生物学活性,达到0.45 U/mg。

Objective: To clone the TK1 cDNA sequence of zabrafish,to express its protein in E.coli and to evaluate its biological activity.Method: The complete TK1 cDNA was amplified by RT-PCR and RACE.Expression vectors were induced by IPTG in E.coli BL21(DE3).TK1 protein was purified to homogeneity using Ni-NTA spin column.Result: The complete TK1 cDNA sequence was cloned,encoding a protein with a molecular weight of 26 kD.Conclusion: The purified zebrafish TK1 showed the highest biological activity,0.45 U/mg at 28℃.