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KMID : 0381120130350010095
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Genes and Genomics
2013 Volume.35 No. 1 p.95 ~ p.100
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Cloning and characterization of hemerythrin gene from Sipuncula Phascolosoma esculenta
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Liu Yan
Li Chenghua
Su Xiurong
Wang Mengqian
Li Yanyan
Li Ye
Li Taiwu
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Abstract
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Hemerythrin is a non-heme respiratory protein involved in oxygen storage and transport in invertebrates. In the present study, the hemerythrin cDNA was cloned from Phascolosoma esculenta (denoted as PeHr) by PCR and rapid amplification of cDNA ends approaches. The full-length PeHr consisted of 770 bp containing of a 5′-terminal untranslated region (UTR) of 83 bp, a 3′-terminal UTR of 327 bp, and a coding domain sequence of 360 bp encoding a polypeptide of 120 amino acids with estimated molecular mass of 13.6 kDa and theoretical isoelectric point of 5.78. The expression profiles of PeHr were evaluated by real-time RT-PCR under blood loss stress. The expression level of PeHr was significantly up-regulated from 45 to 48 h, then slightly recovered to its original level. The coding sequence of the PeHr was cloned and expressed in Escherichia coli BL21 (DE3) for antibodies preparation. Western blotting analysis conformed that the generated antibodies could specifically identify not only recombinant product, but also native protein from the total protein extraction. Our results indicated that PeHr might be involved into haemocytes regeneration, and its function roles should be further investigated by the generated antibodies.
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KEYWORD
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Phascolosoma esculenta, Hemerythrin, Blood loss stress, Western blot
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