文章摘要
王宇,刘菊英,王贤裕,等.蛋白转导结构域4-铜/锌-超氧化物歧化酶融合蛋白的制备及其穿细胞膜的功能研究[J].安徽医药,2018,22(2):220-223.
蛋白转导结构域4-铜/锌-超氧化物歧化酶融合蛋白的制备及其穿细胞膜的功能研究
Preparation of Fusion Protein PTD4-Cu/ZnSOD and verification of its ability to penetrate the cell membrance
投稿时间:2016-12-20  
DOI:
中文关键词: 铜/锌-超氧化物歧化酶  蛋白转导结构域  心肌细胞
英文关键词: 
基金项目:国家自然科学基金项目(81171783)
作者单位E-mail
王宇 十堰市太和医院、湖北医药学院附属医院麻醉科,湖北 十堰 442000  
刘菊英 十堰市太和医院、湖北医药学院附属医院麻醉科,湖北 十堰 442000 wang_yu000@sina.com 
王贤裕 十堰市太和医院、湖北医药学院附属医院麻醉科,湖北 十堰 442000  
柯昌斌 十堰市太和医院、湖北医药学院附属医院麻醉科,湖北 十堰 442000  
李瑞明 十堰市太和医院、湖北医药学院附属医院生物医学研究所,湖北 十堰 442000  
王晓勋 十堰市太和医院、湖北医药学院附属医院生物医学研究所,湖北 十堰 442000  
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中文摘要:
      目的 拟构建蛋白转导结构域(PTD)4-铜/锌-超氧化物歧化酶(Cu/ZnSOD)原核表达质粒,纯化制备PTD4-Cu/ZnSOD融合蛋白,探讨其穿膜能力。 方法 设计合成hCu/ZnSOD引物,扩增hCu/ZnSOD 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PET16b-PTD4进行重组,扩增质粒,双酶切鉴定和DNA测序;获得PET16b-PTD4-Cu/ZnSOD (CDs)原核表达载体,导入感受态E.coli BL21(DE3)中,进行扩增培养,收集菌体,将其裂解离心、Ni+2亲和层析、收集目的蛋白。融合蛋白孵育人心肌细胞,免疫荧光法检测融合蛋白穿膜能力。 结果 通过测序证实实验扩增所得的PTD序列与设计的预期序列一致。设计并简并PTD4-Cu/ZnSOD基因长度为567 bp,通过引物进行正反向测序证实该序列与已被登录的GENBANK中Cu/ZnSOD序列相一致,并构建了相应的原核表达质粒。并在E.coli BL21进行大量表达,最终收集的PTD4-Cu/ZnSOD具有良好水溶性。SDS-page分析显示构建的PTD4-Cu/ZnSOD分子量约为20 kDa,与预期的蛋白分子量20.45 kDa相符合。融合蛋白可以穿透心肌细胞胞膜,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PET16b-PTD4-Cu/ZnSOD原核表达质粒,获得了可穿透心肌细胞膜的PTD4-Cu/ZnSOD融合蛋白,为应用Cu/ZnSOD治疗缺血性疾病的研究奠定了基础。
英文摘要:
      Objective This study is aimed to construct prokaryotic expression plasmid PTD4-Cu/ZnSOD and purify PTD4-Cu/ZnSOD fusion protein,to explore the property of intercellular transport. Methods The primers of hCu/ZnSOD were designed and synthesized to amplify full-length hCu/ZnSOD cDNA by PCR cloning assay.After double-enzyme digestion,the linearized PET16b-PTD4 and hCu/ZnSOD cDNA was ligated with PCR cloning reaction to construct PET16b-PTD4-Cu/ZnSOD.The recombinant plasmid was verified by PCR and DNA sequencing,and transformed into E.coli BL21 (DE3) host bacteria which was induced to obtain fusion protein PTD4-Cu/ZnSOD.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.The transmembrane ability of fusion protein was investigated by examination of PTD4-Cu/ZnSOD in cells using immunofluorescence on cultured cardiomyocytes. Results The sequence of amplified PTD4 was identical with the designed The PTD4-Cu/ZnSOD fusion gene was amplified successfully and the length of thesequence was 567 bp.The PTD4-Cu/ZnSOD (CDs) prokaryotic expression vectors inplasmiwere successfully constructed and inserted into E.coli BL21 to produce alarge quantity of recombinant PTD4-Cu/ZnSOD proteins.The expressed fusion proteinPTD4-Cu/ZnSOD was souble and about 20 kDa which was accroding with the expected recombinant products.The result of immunofluorescence suggested that the fusion protein could transduce into cardiomyocytes with native activity. Conclusion The PTD4-Cu/ZnSOD fusion protein was successfully prepared and could be efficiently transduced into cardiomyocytes with native activity,which provides a basis for the research on treatment and prevention of myocardial ischemia-reperfusion injury with Cu/ZnSOD.
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