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

作　　者：马克学[1] 娄昊[1] 陈广文[1] 刘德增[1] 

机构地区：[1]河南师范大学生命科学学院,新乡453007

出　　处：《动物学杂志》2011年第4期72-77,共6页Chinese Journal of Zoology

基　　金：国家自然科学基金项目(No.30870368;30670247;30170119);河南省杰出青年科学基金项目(No.0312001100);河南省创新人才基金项目(No.2005126);教育部高等学校博士点基金项目(No.200804760003)

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。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.

关 键 词：涡虫 HSP70 原核表达 蛋白纯化 抗血清 

分 类 号：Q78[生物学—分子生物学]