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

Chang-Geng Yang,Bing Sun,Shan-Shan Liu,Bo Zhang,Song-Lin Chen,Xian-Li Wang

doi:10.1186/1471-2199-14-10

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.

Highlights

Biological processes are primarily performed and controlled by proteins
Cloning and sequence analysis of PoAkirin1 The PoAkirin1 cDNA acquired from the full-length cDNA Library of Japanese flounder comprised a 5′UTR of 202 bp, an open reading frames (ORFs) of 564 bp encoding a polypeptide of amino acids and a 521-bp 3′UTR with a poly (A) tail (Figure 2)
The results showed that the yeast grew with a blue color in the yeast rotary verification test using PoC1q and PoAkirin1, and the yeast expressing PoHEPN with a 96–223 aa segment deleted and PoAkirin with a 146–223 aa segment deleted were blue when grown on SD/–Leu/–Trp/x-α-gal. (Figures 1, 9, and 10)

Summary

Introduction

Biological processes are primarily performed and controlled by proteins. clarifying the biological functions of proteins and their biological response mechanisms at the cellular level has become the main objective of proteomics research. NF-κB can be activated by a variety of stimulatory factors, including cytokines, lymphokines, UV, pharmaceutical preparations, and growth and stress factors Such activation of NF-κB is part of the stress response. Many members in the NF-κB signaling pathway have been identified in the past 20 years, a highly conserved protein Akirin, a member in the NF-κB signaling pathway, was recently identified in a study of the immune defense system at 2008 [8]. This 20–25-kDa protein participates in the regulation of gene expression in

Methods

Results

Conclusion