日本三角涡虫hsp70 cDNA克隆及原核表达

马克学; 陈广文; 娄昊; 费利娜

日本三角涡虫hsp70 cDNA克隆及原核表达

基金项目:

国家自然科学基金(No.30870368,30670247,30170119)

河南省杰出青年基金(No.0312001100)

河南省创新人才基金(No.2005126)

教育部高等学校博士点基金(No.200804760003)资助

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出版历程
- 收稿日期: 2009-03-11
- 修回日期: 2009-11-21
- 发布日期: 2010-03-24

CLONING AND PROKARYOTIC EXPRESSION OF HSP70 cDNA FROM PLANARIAN DUGESIA JAPONICA

摘要

摘要: 热休克蛋白70 是热休克蛋白家族中的重要成员, 它在保护生物体免受各种胁迫中发挥重要作用。由于其惊人的再生能力, 淡水涡虫作为研究再生和发育的模式动物受到研究者关注。但是, 有关涡虫抗逆性的分子机制却少有报道。研究采用 RACE (Rapid amplification of cDNA end) 技术首次从日本三角涡虫中克隆出hsp70 (Djhsp70)全长cDNA 序列。Djhsp70 cDNA 全长2066 bp, 含有1947 bp 的开放阅读框, 编码648 个氨基酸, 分子量71.18 kD, GenBank 登录号EU380241。DjHSP70 的氨基酸含有真核生物HSP70 家族蛋白的三个标签序列(9–16 位的IDLGTTYS、199—206 位的 DLGGGTFD、334–339 位的IVLVGG)和末端高度保守序列EEVD。经BLAST 检索分析, Djhsp70 的核苷酸序列和推定的氨基酸序列与目前已知HSP70 家族成员高度同源。有趣的是, HSP70 亲缘关系分析表明: 涡虫更靠近脊椎动物, 而与无脊椎动物果蝇和线虫相距较远。为了制备抗体研究DjHSP70 的组织学定位, 实验还成功构建了DjHSP70 表达载体, 在IPTG 诱导下表达出约76 kD 的融合蛋白。Djhsp70 cDNA 的克隆与表达载体的构建为下一步工作奠定了基础。
- 日本三角涡虫 /
- HSP70 /
- 克隆 /
- 表达
Abstract: Heat shock protein 70 (HSP70) is an important member of the heat shock protein family, and it plays a key role in the process of protecting organisms from various stresses. Owing to its powerful regenerating ability, freshwater planarian has been attached high importance as model animal for the study of development and regeneration. However, little reports has addressed on stress response in planarians. In this study, the full-length hsp70 cDNA was firstly isolated and sequenced from planarian Dugesia japonica (Djhsp70) using rapid amplification of cDNA end (RACE) technology. The full-length cDNA of Djhsp70 was 2066 bp containing an open reading frame (ORF) of 1947 bp encoding a polypeptide of 648 amino acids with a predicted molecular mass of 71.18 kDa, which was registered in GenBank with accession No. EU380241. The deduced amino acid sequence of DjHSP70 shared three signatures motifs (IDLGTTYS at the position of 9—16, DLGGGTFD at 199—206 and IVLVGG at 334—339) and the C-terminal consensus EEVD of eukaryotic HSP70 family. A sequence homology search using BLASTN and BLASTP revealed that DjHSP70 had high homology with other known HSP70s. Interestingly, the phylogenetic analysis of HSP70 revealed that planarian had a closer relationship to vertebrates, but far from to other invertebrates such as D. melanogaster and C. elegans. For preparing the antibody to study the subcellular location of DjHSP70, DjHSP70 expression vector was successfully constructed and expressed a 76 kDa fusion protein in agreement with the expected molecular weight after the induction with IPTG. Djhsp70 cDNA cloning and construction of its expression vector provided a basis for further study in our laboratory.
- Planarian Dugesia japonica /
- HSP70 /
- Cloning /
- Expression

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参考文献(19)

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