天津医药

• 细胞与分子生物学 • 上一篇    下一篇

人源性防御素HD-5的原核表达及抗真菌作用的初步研究

张萍萍1,尹利荣1,2,王芳1,孙蓓2,3   

  1. 1. 天津医科大学第二医院
    2.
    3. 天津医科大学内分泌研究所
  • 收稿日期:2013-09-25 修回日期:2014-03-28 出版日期:2014-08-15 发布日期:2014-08-15
  • 通讯作者: 张萍萍

Prokaryotic Expression and Antifungal Activity of Human α defensin-5 Protein

  • Received:2013-09-25 Revised:2014-03-28 Published:2014-08-15 Online:2014-08-15
  • Contact: Ping-Ping ZHANG

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摘要：

【摘要】目的  构建pQE-30Xa/HD-5原核表达载体，纯化重组蛋白并进行抗真菌活性的初步鉴定。方法  以pcDNA3.1(+)/HD-5为模板，聚合酶链反应（PCR）扩增编码HD-5成熟肽的基因。构建pQE-30Xa/HD-5重组表达载体，并对重组质粒进行酶切、基因序列分析。将鉴定正确的质粒转化入大肠杆菌M15后进行异丙基-D-硫代半乳糖苷(IPTG)诱导表达，对表达产物进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定。通过镍柱亲和层析纯化蛋白并进行蛋白复性，蛋白免疫印迹（Western blot）鉴定纯化产物。以KB纸片法初步验证纯化获得的重组蛋白对白色假丝酵母菌的抑菌活性。结果  成功克隆了HD-5基因并构建了重组质粒pQE-30Xa/HD-5。重组质粒在大肠杆菌M15中诱导表达出HD-5融合蛋白；Western blot分析结果显示纯化后的融合蛋白与目的蛋白相符；KB纸片法证实纯化后的融合蛋白对白色假丝酵母菌具有一定的抑菌活性。结论  成功构建HD-5原核表达载体，经诱导表达后纯化获得具有良好抑菌活性的HD-5融合蛋白。

关键词: 防御素类, 重组融合蛋白质类, 大肠杆菌, 防御素-5, 原核表达, 亲和层析, 抗真菌活性

Abstract: 　　[Abstract] Objective   To construct the prokaryotic expression vector for HD-5and purify the recombinant HD-5 protein then analyze its antifungal activity.Methods   The HD-5gene was cloned by PCR, then was inserted into prokary?otic expression plasmid pQE-30Xa to construct pQE-30Xa/HD-5. After sequencing, pQE-30Xa/HD-5 gene was transformed in?toE.coliM15cells. Its expression was induced by IPTG and confirmed by SDS-PAGE. The recombinant protein was purified through Ni-NTA affinity purification system. The antifungal activity was tested by disk diffusion method. Results   HD-5 gene and pQE-30Xa/HD-5vector were obtained successfully.E.coli M15strains was used to express HD-5fusion protein.After purification, the fusion protein was confirmed by Western blot. The disk diffusion test confirmed that the fusion pro?tein can inhibit Candida albicans. Conclusion   Expression vector pQE-30Xa/HD-5was successfully constructed. The HD-5fusion protein was expressed inE.colisuccessfully, which showed a certain degree of antifungal activity.

Key words: defensins, recombinant fusion proteins, Escherichia coli, Human alpha defensin 5, prokaryotic expression, affinity purification, antifungal activity