中文摘要：

目的：制备血红素加氧酶-1（HO-1）融合蛋白PEP-1-HO-1,观察融合蛋白在大鼠H9c2心肌细胞的转导。方法：设计合成hHO-1 PCR引物,扩增hHO-1 cDNA片段;采用PCR靶向克隆法将扩增产物与含有相同酶切位点载体质粒pET15b-PEP-1进行重组,扩增质粒,双酶切鉴定和DNA测序;将测序和鉴定结果正确的重组质粒转化宿主菌Rosetta（DE3）pLysS,诱导表达融合蛋白PEP-1-HO-1,利用表达蛋白N端的组氨酸＂标签＂（His-tag）进行亲和层析纯化融合蛋白;SDS-PAGE和Western blotting对融合蛋白进行定性检测,Bradford法定量检测纯化后蛋白;融合蛋白孵育大鼠H9c2心肌细胞,免疫组化法观察融合蛋白在H9c2细胞的转导和分布,分光度法检测蛋白转导后的活性。结果：hHO-1 cDNA与pET15b-PEP-1重组成功,重组质粒pET15b-PEP-1-hHO-1经酶切鉴定含有hHO-1 cDNA,测序分析证实与GenBank提供的原始序列完全一致;重组质粒转化后经诱导和纯化得到了融合蛋白PEP-1-HO-1,浓度达到1.0 g/L;融合蛋白可以穿透H9c2心肌细胞胞膜,进入细胞后主要分布在胞质和胞核内并具有天然活性。结论：成功地构建了pET15b-PEP-1-hHO-1原核表达质粒,获得了可穿透H9c2心肌细胞膜的血红素加氧酶-1融合蛋白PEP-1-HO-1,为应用HO-1治疗缺血性疾病的研究奠定了基础。

英文摘要：

Objective： To prepare the HO-1 fusion protein PEP-1-HO-1 and transduct it into rat myocardial H9c2 cells.Methods： The primers of hHO-1 were designed and synthesized to amplify full-length hHO-1 cDNA by PCR cloning.After double-enzyme digestion,the linearized pET15b-PEP-1 and hHO1 cDNA was ligated with PCR cloning reaction to construct pET15b-PEP-1-hHO-1.The recombinant plasmid was verified by PCR and DNA sequencing,and transformed into Rosetta（DE3）pLysS host bacteria which was induced to obtain fusion protein PEP-1-HO-1.The fusion protein was purified with affinity chromatography because of the recombinant protein having an N-terminal His-tag sequence and characterized by SDS-PAGE and Western blot.Bradford method was performed to quantify the purified PEP-1-HO-1 fusion protein.The distribution of transduced fusion protein in H9c2 cardiomyocytes was evaluated by immunofluorescence,and the activity of fusion protein in cells was detected by spectrophotometric method.Results： hHO-1 cDNA and pET15b-PEP-1 was ligated successfully.The restriction endonuclease analysis demonstrated pET15b-PEP-1-hHO-1 contained human full length hHO-1 cDNA.SDS-PAGE and Western blot demonstrated the fusion protein could successfully expressed by Rosetta（DE3）pLysS host bacteria and it was purified with affinity chromatography.The result of immunofluorescence suggested that the fusion protein could be transduced into H9c2 cardiomyocytes with native activity.Conclusion： The PEP-1-HO-1 fusion protein was successfully prepared and could be efficiently transduced into H9c2 cardiomyocytes with native activity,and it provides a basis for the research on treatment and prevention of myocardial ischemia-reperfusion injury with HO-1.