中文摘要：

鸢尾素（Irisin）是新发现的一种肌肉因子,其前体是Ⅲ型纤连蛋白组件包含蛋白5（fibronectin typeⅢdomain-containing protein 5,FNDC5）。为实现人（Homo sapiens）Irisin的原核表达,通过反转录PCR得到人肌肉组织总RNA的c DNA,并以特异性引物扩增得到人FNDC5基因的c DNA序列。以特异性引物扩增将人Irisin基因序列引入NdeⅠ和XhoⅠ酶切位点,经双酶切后插入原核表达载体p ET-30a中,构建重组质粒并转化大肠杆菌（Escherichia coli）Rosetta（DE3）p Lys S。通过异丙基-β-D-硫代吡喃半乳糖苷（isopropylβ-D-1-thiogalactopyranoside,IPTG）诱导表达,进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl dulfate polyacrylamide gel electropheresis,SDS-PAGE）检测,用镍柱纯化目的蛋白,并用Western blot方法鉴定。DNA序列分析结果显示,克隆的人Irisin基因序列和NCBI公布的序列一致,并成功构建了p ET-30a-Irisin重组表达载体;SDS-PAGE电泳检测和Western blot结果表明,表达蛋白分子量约为14 k D,与预期大小一致;实现了人Irisin的可溶性表达,得到了较高纯度的目的蛋白,为进一步研究人Irisin蛋白的结构、功能及应用提供了理论依据。

英文摘要：

As a recently discovered muscle factor, human（Homo sapiens） Irisin is an excretive polypeptide fragment whose precursor is fibronectin type Ⅲ domain- containing protein5（FNDC5）. Irisin can transfer the signal of skeletal muscle and keep the relationship between skeletal muscle and the peripheral tissues. Irisin will certainly be a very promising active factor, and become the new targets for prevention and control of metabolic disease and its complications. Therefore, exploiting a method that can be used to mass produce and easily purify human Irisin recombinant protein is very significant. To achieve the prokaryotic expression of human Irisin, the FNDC5 c DNA was obtained from the muscles, then the restriction enzyme cutting site NdeⅠand XhoⅠ were introduced to human Irisin gene by the specific primers amplification. The gene sequence was digested by NdeⅠ and XhoⅠ and cloned into prokaryotic expression vector, p ET-30 a that digested by NdeⅠand XhoⅠ, to form p ET-30a-Irisin. The recombinant plasmid was transformed to Escherichia coli Rosetta（DE3）p Lys S and was screened by kanamycin to form Rosetta- p ET- 30a- Irisin recombination strain. Then the expression of interest protein was induced with 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L isopropyl β- D- 1-thiogalactopyranoside（IPTG）, and the recombinant protein detected by sodium dodecyl sulfate polyacrylamide gel electropheresis（SDS- PAGE）, the gommures was stained by Coomassie brilliant blue for 4 hours and discolored for 3 hours to analyze the expression of recombinant proteins. In order to realize the soluble expression of recombinant proteins, the Rosetta- p ET- 30a- Irisin recombination strain was induced with 0.1mmol/L IPTG at 18 ℃. And the recombination strains induced with IPTG were broken by ultrasonication, the soluble recombinant proteins in cracked supernatant were purified by Ni- sepharose, then detected and identified by Western blot. The DNA sequence analysis showed, that the sequence of cloned human Irisin was i