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Molecular and immune response characterizations of IL-6 in large yellow croaker (Larimichthys crocea)

Zhu, Qian, Li, Chan, Yu, Zhen-Xing, Zou, Peng-Fei, Meng, Qing-Xiang, Yao, Cui-Luan
Fish & shellfish immunology 2016 v.50 pp. 263-273
phylogeny, signal transduction, fish, mammals, blood, polypeptides, non-specific protein-tyrosine kinase, open reading frames, amino acids, spleen, Vibrio parahaemolyticus, signal peptide, stomach, introns, tissues, Escherichia coli, messenger RNA, complementary DNA, gene overexpression, Larimichthys crocea, transcription (genetics), tumor necrosis factor-alpha, immune response, kidneys, exons, interleukin-1beta, quantitative polymerase chain reaction, liver, inflammation, interleukin-6, reverse transcriptase polymerase chain reaction
Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine which exists in multiple tissues and cell lines. In the present study, the full-length cDNA and the genomic sequence of IL-6 (LcIL-6) were cloned from large yellow croaker, Larimichthys crocea. The full-length cDNA of LcIL-6 was 1066 base pairs (bp), containing an open reading frame (ORF) of 678 bp encoding for 225 amino acids, a 5′ untranslated region (UTR) of 71 bp and a 3′ UTR of 317 bp. The predicted LcIL-6 protein included a 24 amino acids (aa) signal peptide and a conserved IL-6 domain. However, the polypeptide sequence identities between LcIL-6 and its counterparts in mammals and other fish are from 12% to 45%. The genome sequence of LcIL-6 gene was composed of 2126 bp, including five exons and four introns. Phylogenetic analysis revealed that LcIL-6 showed a close relationship with the IL-6 from other bony fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that LcIL-6 mRNA was expressed in most examined tissues, with the most predominant expression in stomach, followed by blood and very weak expression in other tissues. The expression levels of LcIL-6 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were investigated in spleen, head-kidney and liver. LcIL-6 transcripts were induced significantly after immune challenge, with the peak-value of 33.5 times as much as the control in the head-kidney at 3 h after LPS injection (p < 0.05). Overexpression of LcIL-6 enhanced tumor necrosis factor (TNF)-α transcripts significantly (p < 0.05) in L. crocea kidney (LCK) cells. Additionally, recombinant LcIL-6 mature peptide was obtained in the supernatant of Escherichia coli BL21 (DE3). The purified recombinant LcIL-6 fusion protein was also demonstrated to improve the transcriptional expression levels of TNF-α significantly in LCK cells (p < 0.05). However, no significant changes of Mx (myxovirus resistant protein), IL-1β, janus kinase (JAK)2, signal transducers and activators of transcription (STAT)3 and STAT5 in LCK cells was detected after LcIL-6 overexpression or recombinant LcIL-6 protein stimulation. Our results indicated that LcIL-6 might be important in large yellow croaker immune response and improve the inflammatory response by through activation TNF-α expression.