Cloning of common carp SOCS-3 gene and its expression during embryogenesis, GH-transgene and viral infection.

As a member of a newly discovered protein family, the suppressor of cytokine signalling 3 (SOCS-3) has been shown to regulate the responses of many immune cytokines in a negative auto-regulatory manner. The full-length cDNA of common carp SOCS-3 was 1603 bp and contained a 630 bp open reading frame (ORF) coding for a protein of 209 amino acids. Carp SOCS-3 molecule was well conserved especially in the SRC homology 2 (SH2) and the SOCS box. The kinase inhibitory region (KIR) and ESS domains, upstream of the SH2 domain, were conserved in carp SOCS-3, except for a specific insertion (PHRYK) in the KIR domain at the N-terminal region. Three conserved cysteine (Cys-102, 124 and 193) residues, and one additional cysteine (Cys-168) residue, were also found in carp SOCS-3. The 2015 bp genomic DNA of carp SOCS-3 contained two exons and one intron. Phylogenetic analysis showed that carp SOCS-3 sequence grouped with other known fish SOCS-3 sequences with zebrafish SOCS-3 as the closest neighbour. RT-PCR analysis showed that carp SOCS-3 was initially expressed at 4 h pf (post-fertilization) and gradually increased up to 4 w pf during embryogenesis. By RT-qPCR analysis, carp SOCS-3 gene was predominantly detected in gill, head kidney, thymus and skin, followed by spleen and peripheral blood, lower expression level was detected in kidney, intestine, liver and muscle; the SOCS-3 transcript was significantly increased in thymus, head kidney, spleen and intestine of GH (growth hormone)-transgenic carp; after SVCV (spring viraemia of carp virus) infection, the carp SOCS-3 transcript was significantly up-regulated in gill, intestine, thymus, spleen, head kidney and kidney tissues in a time-dependent manner. These results suggest that teleost SOCS-3 may play an active role in the modulation of viral-induced innate immune response and in preventing the overaction of some cytokines with viral stimulation.

Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene.

Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp 3'-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1-k long genomic DNA of carp NKEF-B containing six exons and five introns. Real-time RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene.

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5'-terminal untranslated region (UTR) of 285 bp, a 3'-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.

Molecular cloning and characterization of common carp (Cyprinus carpio L.) TCRgamma and CD3gamma/delta chains.

Partial cDNA sequences of TCRgamma and CD3gamma/delta were isolated from the thymus of common carp (Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCRgamma and CD3gamma/delta were obtained by means of 3' RACE and 5' RACE, respectively. The full length of carp TCRgamma chain is 1368bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCRgamma contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCRgamma. The C region of carp TCRgamma contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR Cgamma contains 37 amino acids. The full length of carp CD3gamma/delta is 790bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3gamma/deltas, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3gamma/delta. Differing from other known CD3gamma/deltas, carp CD3gamma/delta lacks the CXXCXE motif in the extracellular domain. RT-PCR analysis demonstrated that the expression of TCRgamma gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCRgamma gene was detected in all the examined tissues. The expression of CD3gamma/delta gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3gamma/delta-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone.

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8beta and CD4-like genes.

Partial cDNA sequences of both CD8beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8beta and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8beta is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8betas, carp CD8beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp.