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

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...

Expression of the C-terminal Polypeptide of Planarian Heat Shock Protein 70 and Preparation of Its Polyclonal Antibody

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.