| 马克学,娄昊,陈广文,刘德增.2011.日本三角涡虫热休克蛋白70(DjHSP70)C-端多肽表达及其抗血清制备.动物学杂志,46(4):72-77. |
| 日本三角涡虫热休克蛋白70(DjHSP70)C-端多肽表达及其抗血清制备 |
| Expression of the C-terminal Polypeptide of Planarian Heat Shock Protein 70 and Preparation of Its Polyclonal Antibody |
| 投稿时间：2011-01-22 修订日期：2011-04-10 |
| DOI： |
| 中文关键词: 涡虫 HSP70 原核表达 蛋白纯化 抗血清 |
| 英文关键词:Planarian HSP70 Prokaryotic expression Protein purification Anti-serum |
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| 中文摘要: |
| 首先运用在线生物学软件对日本三角涡虫(Degusia japonica)热休克蛋白70(DjHSP70)氨基酸序列进行亲水区分析,发现该蛋白C-端含有较多亲水性氨基酸,然后以该段多肽序列为基础构建原核表达载体。采用PCR方法扩增450 bp cDNA片段,编码DjHSP70 C-端150个氨基酸多肽。将双酶切的cDNA片段与pET-28a载体连接后导入BL21受体菌,在IPTG诱导下表达出21 ku融合蛋白,分子量与预期相符。该融合蛋白采用Ni2+-NTA agarose树脂进行纯化,纯化结果电泳检测后经灰度扫描分析显示纯度在95%以上。融合蛋白免疫新西兰大白兔获得高效价的抗血清,Western blot检测显示该抗血清不仅具有很强的特异性,而且还识别小鼠HSP70。此项工作为进一步研究淡水涡虫抗逆性奠定了基础。 |
| 英文摘要: |
| In this paper, hydrophilic domain of DjHSP70 was predicted by using internet biosoftware. The DjHSP70 C-terminal contains numerous hydrophilic amino acids. Based on this hydrophilic domain of DjHSP70, the prokaryotic expression vector was constructed. PCR method was used to amplify 450 bp cDNA fragment encoding DjHSP70 C-terminal 150 amino acid polypeptides. After digested by Hind Ⅲ/XhoⅠ, this cDNA fragment was ligated to pET-28a expression vector. Recombinant plasmid was transformed into E.coli BL21 and a 21 ku fusion protein was expressed after the induction with IPTG, which was in agreement with the expected molecular weight. This fusion protein was purified using Ni2+-NTA agarose and detected by SDS-PAGE electrophoresis. Grayscale scanning showed that the purity of the purified protein was over 95%. The fusion protein was used as an antigen to immunize New Zealand rabbits to prepare the polyclonal antibody. The results showed that this anti-serum was not only very specific to DjHSP70, but also recognized mouse HSP70.This work has laid the foundation for further investigating stress responses in freshwater planarians. |
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