中文摘要：

目的：构建昆明小鼠十二指肠肠激酶轻链（mEKL）大肠杆菌高效表达系统，并建立复性与纯化方法。方法：RT—PCR法扩增mEKL全长cDNA，以融合表达载体pET32a克隆于大肠杆菌BL21（DE3）和Origami（DE3），采用阴离子交换层析纯化复性表达产物，经牛肠激酶消化回收mEKL。结果：扩增得到723bp的mEKL全长cDNA，经序列分析发现，昆明鼠来源的mEKL cDNA序列的Ser^131密码子中存在1个同义点突变。mEKL在2种宿主菌中的表达产物均为包涵体蛋白，经稀释复性、阴离子交换层析纯化和肠激酶切割可得高纯度mEKL。结论：成功构建昆明鼠源mEKL高效表达工程菌，并建立相应的纯化方法。

英文摘要：

Objective：To construct recombinant Escherichia bacteria over - expressing the light chain of entcrokinase from Kunming mice （ mEKL ） and develop a purification procedure of the expression product. Method ： The full - length cDNA of mEKL was amplified by RT - PCR, and cloned into E. coli BL21 （DE3） and Origami （DE3） in pET32a for fusion -expressing with thioredoxin（Trx）under the control of promoter T7. The expression product was purified by anion exchange chromatography （AEC）after refolding, from which mEKL was recov- ered by enterokinase digestion. Result： A 723 bp cDNA coding mEKL was from Kunming mice＇ s duodenum,in which a synonymous point mutation was found in the codon of Ser^131. The fusion gene of Trx - mEKL was over - expressed as inclusion proteins in both host cells. The refolded fusion protein was purified by AEC to an over 80% purity,from which mEKL was recovered by enterokinase digestion and can be further purified by Ni - chelating affinity chromatography. Conclusion：An over - expression system for the recombinant mEKL was established,Which laid a good foundation for the over- production of enterokinase.