CLONING AND PROKARYOTIC EXPRESSION OF HSP70 cDNA FROM PLANARIAN DUGESIA JAPONICA

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.