中文摘要：

目的利用原核生物表达载体表达小鼠铁调素功能肽Hepc25，并对其进行纯化。方法选取编码25aa的Hepc25基因与硫氧还蛋白基因融合后在大肠杆菌中大规模诱导表达该融合蛋白，以RT—PCR法扩增小鼠Hepcidinl功能肽基因Hepc25，将目的基因克隆到原核表达载体pET-32a（＋）中获得pET．32a—Hepc25，再以pET-32a-Hepc25转化大肠杆菌BL21表达融合蛋白，收集菌体蛋白，用Westernblotting和SDS—PAGE法检测融合蛋白，优化融合蛋白高表达条件。依次利用亲和层析、离子交换层析和凝胶排阻层析法纯化融合蛋白，融合蛋白经肠激酶酶切、高效液相色谱法（HPLC）分离纯化获得了目的蛋白Hepc25。结果成功构建了小鼠pET-32a—Hepc25融合蛋白表达载体，融合蛋白表达量约占大肠杆菌BL21总蛋白量的28％，融合蛋白在33℃、120r／min、终浓度为0．2mmol／L的异丙基-B-D硫化半乳庶糖（IPTG）诱导8h的条件下表达量最高，融合蛋白纯度达95％。融合蛋白经过色谱分离、酶切和HPLC分析，目的蛋白的纯度也达95％以上。结论利用原核生物表达载体成功表达并纯化了小鼠铁调素功能肽Hepc25，并筛选出了高表达的优化条件。

英文摘要：

Objective To purify functional peptide Hepc25 of mouse and examine its expression by using the expression vector of prokaryote. Methods The gene of Hepc25 was fused with thioredoxin gene and the fusion protein was expressed greatly in E. eiol. Hepe25 gene amplification was done by RT-PCR and the gene was cloned into pET32a（ ＋ ） plasmids. The pET-32a-Hepc25 plasmids were transformed into E. ciol BL21 competent cells. In order to obtain much of the fusion protein, the induction condition was optimized. The fusion protein was measured by SDS-PAGE and Western blotting. Isolation and purification of fusion protein were done by affinity chromatograph, iron exchange chromatography and exclusion chromatography. Purified Hepc25 from fusion protein hydrolyzed by enterokinase was analyzed by HPLC. Results Fusion protein expression vectors were constructed successfully and the quantity of fusion protein was about 28% of total protein. Under the condition of 33~C , 120 r/min,0. 2 mmol/L isopropyl β-D-thiogalactopy ranoside（IPTG） for 8 hours,the quality of expression of fusion protein was the largest and the purity of fusion protein and Hepc25 were both over 95%. Conclusion Functional peptide Hepc25 of mouse is expressed by using prokaryotic expression vector and is purified. The optima expression condition is choosed.