Cloning and expression of HSP70 gene of sipuncula Phascolosoma esculenta.
In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5'-terminal untranslated region (UTR) (125 bp), a 3'-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for 12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 degrees C for 24 h sea water.
Su
Xiurong
X
Faculty of Life Science and Biotechnology, Ningbo University, Fenghua Road 818, Ningbo City, Zhejiang Province 315211, PR China. suxiurong@gmail.com
Du
Lili
L
Li
Yanyan
Y
Li
Ye
Y
Zhou
Jun
J
Li
Taiwu
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D019216:Metals, Heavy / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009348:Nematoda / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny
D018840:HSP70 Heat-Shock Proteins; D019216:Metals, Heavy