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Open Access Research article

Screening and analysis of PoAkirin1 and two related genes in response to immunological stimulants in the Japanese flounder (Paralichthys olivaceus)

Chang-Geng Yang12, Xian-Li Wang3, Bo Zhang4, Bing Sun1, Shan-Shan Liu1 and Song-Lin Chen1*

Author Affiliations

1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Qingdao 266071, China

2 Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China

3 Translational Center for Stem Cell Research, Tongji Hospital, Stem Cell Research Center, Tongji University School of Medicine, Shanghai 200065, China

4 Bohai Sea Fisheries Research Institute of Tianjin, Tianjin, China

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BMC Molecular Biology 2013, 14:10  doi:10.1186/1471-2199-14-10

Published: 7 May 2013

Abstract

A member of the NF-κB signaling pathway, PoAkirin1, was cloned from a full-length cDNA library of Japanese flounder (Paralichthys olivaceus). The full-length cDNA comprises a 5′UTR of 202 bp, an open reading frame of 564 bp encoding a 187-amino-acid polypeptide and a 521-bp 3′UTR with a poly (A) tail. The putative protein has a predicted molecular mass of 21 kDa and an isoelectric point (pI) of 9.22. Amino acid sequence alignments showed that PoAkirin1 was 99% identical to the Scophthalmus maximus Akirin protein (ADK27484). Yeast two-hybrid assays identified two proteins that interact with PoAkirin1: PoHEPN and PoC1q. The cDNA sequences of PoHEPN and PoC1q are 672 bp and 528 bp, respectively. Real-time quantitative reverse-transcriptase polymerase chain reaction analysis showed that bacteria could induce the expressions of PoAkirin1, PoHEPN and PoC1q. However, the responses of PoHEPN and PoC1q to the bacterial challenge were slower than that of PoAkirin1. To further study the function of PoAkirin1, recombinant PoAkirin1 and PoHEPN were expressed in Escherichia coli and would be used to verify the PoAkirin1-PoHEPN binding activity. These results identified two proteins that potentially interact with PoAkirin1 and that bacteria could induce their expression.

Keywords:
Akirin; Japanese flounder; NF-κB; Yeast two-hybrid assay; Immunity; HEPN; C1q