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文章摘要
何明旺,项雅婧,孙云,曹贞洁,周永灿.卵形鲳鲹Hepcidin-5的原核表达、纯化及多克隆抗体的制备[J].海南大学学报编辑部:自然科学版,2019,37(1):.
卵形鲳鲹Hepcidin-5的原核表达、纯化及多克隆抗体的制备
Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Hepcidin-5 from Golden Pompano (Trachinotus ovatus)
投稿时间:2018-11-25  修订日期:2018-12-28
DOI:10.15886/j.cnki.hdxbzkb.2019.0006
中文关键词: 卵形鲳鲹;hepcidin 5;原核表达;多克隆抗体
英文关键词: Trachinotus ovatus; hepcidin-5; prokaryotic Expression; polyclonal antibody
基金项目:海南省重大项目(ZDKJ2016011);海南省博士后科学基金(BSH-RST-2018001)
作者单位E-mail
何明旺 海南大学海洋学院 792946791@qq.com 
项雅婧 海南大学海洋学院  
孙云 海南大学海洋学院  
曹贞洁 海南大学海洋学院 1057319886@qq.com 
周永灿 海南大学海洋学院  
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中文摘要:
      为了制备高效的卵形鲳鲹hepcidin 5蛋白多克隆抗体和对其特异性进行鉴定,本研究通过PCR扩增的方法获得了卵形鲳鲹抗菌肽hepcidin 5(TroH5)的成熟肽cDNA序列,并构建了原核表达载体pET 32a(+)/TroH5,然后将其转入大肠杆菌表达菌株BL21(DE3)中进行原核表达.经IPTG诱导,成功表达了相对分子质量约为35kDa的融合重组蛋白.经不同诱导条件的优化,最终确定在IPTG终浓度为0.1mmol/L和温度为20 ℃时,其表达量最大.可溶性分析表明:该融合蛋白主要存在于上清中.经镍柱亲和层析纯化获得了纯度较高的融合蛋白,然后以免疫小鼠制备多克隆抗体,经ELISA法检测,其效价达到1∶105.蛋白免疫印迹(Western blot)检测表明:所制备的多克隆抗体特异性强.本研究为进一步探究hepcidin 5的生物学功能奠定了基础.
英文摘要:
      In order to prepare the efficient anti-hepcidin-5 polyclonal antibody of golden pompano and identify its specificity, this study cloned the sequence that encoded the mature peptide of hepcidin-5 in golden pompano (Trachinotus ovatus). Then, we constructed the recombinant vectors of pET-32a(+)/TroH5 and then transformed into Escherichia coli BL21 (DE3) for the expression. The fusion recombinant protein was successfully induced by IPTG and the putative molecular weight was 28kDa as expected. Solubility analysis showed that the fusion recombinant protein was mainly expressed in the supernatant. Then, after purified by Ni SepharoseTM affinity chromatography column, the fusion recombinant protein with high purity was obtained. The purified fusion recombinant protein was then used to immunize mouse to prepare polyclonal antibody. The polyclonal antibody titer was validated to be 1:105 by ELISA. Western blot analysis showed that the polyclonal antibody was specific. This study will provide the foundation for further exploring the biological functions of hepcidin-5.
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