Molecular And Functional Characterization Of A Novel CXC Chemokine And Immunoglobulin Heavy Chain And Light Chain Isotypes In Large Yellow Croaker

Large yellow croaker(Pseudosciana crocea),is a species of jewfish and is found mainly in the coast in the temperate zone.It is an economically important marine fish species in China, and also represents the largest yield for a single species in Chinese marine net-cage farming. In recent years,with the rapid development of large yellow croaker culture industry,the infectious diseases caused by viruses,bacteria,and parasites are becoming more and more severe,resulting in great economical losses.In the previous study,One EST showing significant homology to CXC chemokines in other species,was obtained from the spleen cDNA library of large yellow croaker by EST analysis in our laboratory.In the current study,we clone and characterize a novel CXCL13-like chemokine gene homologue from large yellow croaker Pseudosciaena crocea (LycCXCL13).The complete cDNA of LycCXCL13 is 796 nucleotides(nt) encoding a protein of 97 amino acids(aa),with a putative molecular weight of 10.7 kDa.The deduced LycCXCL13 contains a 24-aa signal peptide and a 73- aa mature polypeptide,which possesses the typical arrangement of four cysteines as found in other known CXC chemokines(C²⁵,C²⁷,C⁵² and C⁶⁸).It shares 41-44%and 26-40%aa sequence identities to known CXCL13 chemokines in fish species and other vertebrates,respectively.Phylogenetic analysis showed that LycCXCL13 was more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups.Recombinant LycCXCL13 protein produced in E.coli BL21 exhibited marked chemotaxis to the peripheral blood leucocytes(PBLs) from large yellow croaker.LycCXCL13 gene was constitutively expressed in all tissues examined,such as gills,liver,kidney,heart,spleen,muscle and blood,except intestine.Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine,LycCXC13 gene expression was significantly up-regulated in gills,liver,kidney and spleen at 24h after stimulation,and bacterial vaccine was more potent than poly(I:C) in up-regulating LycCXCL13 expression in most of these tissues.Time course analysis using real-time PCR showed that LycCXCL13 gene expression in spleen and kidney was quickly up-regulated by either poly(I:C) or bacterial vaccine,and reached the peak level at 12 h(in both spleen and kidney) post-induction by poly(I:C) and at 24h(in kidney) or 48h(in spleen) post-induction by bacterial vaccine,respectively.These results suggest that LycCXCL13 may have a role in inflammatory response.Imrnunoglobulins(Igs),the most important humoral immune molecule in adaptive immune system,can specifically recognize and bind to the antigen,activate the complement system as well as to mediate different immune response.The cDNA of a immunoglobulin M heavy chains in large yellow croaker(LycIgH) is 1987 nucleotides(nt) encoding a protein of 585 amino acids(aa),with a putative molecular weight of 64.5 kDa.It shares 35.0-61.0%aa sequence identities to known IgHs in fish species.RT-PCR analysis showed that the LycIgH gene was constitutively expressed in varioustissues examined such as gills,intestine,liver, kidney,heart,spleen and blood,while at a higher level in spleen,kidney and intestine.Upon stimulation with poly(I:C),the LycIgH transcripts were quickly increased in spleen and kidney at 12h post induction(with 5.87- and 5.48-fold mRNA increases,respectively), followed by a recovery to normal level at 24h.The LycIgH transcripts in spleen and kidney induced by inactivated bacterial vaccine reached their peak levels at 48h(14.53-fold) and 12h(3.70-fold),respectively.These results indicated the up-regulation of LycIgH expression in spleen and kidney by poly(I:C) or bacterial vaccine occurred at the early phase of induction and was differentially modulated in the two tissues by different stimulation.It might also be the result of NF-kB activation induced by bacterial vaccine or poly(I:C).We also simulate the homology modeling of LycIgH of large yellow croaker.It contains two domains,different from four domains in human IgH.Several light chains were cloned from large yellow croaker by expressed sequence tags (EST) and RACE-PCR techniques.These 10 different clones were grouped to 3 different isotypes.The results of RT-PCR indicated that LycIgL1 and LycIgL2 were detected in various tissue including spleen,kidney,liver,gills,intestine,heart,brain,blood and muscle, except the IgL isotype 3 in heart.After stimulation with poly(I:C) or bacterial vaccine in spleen and kidney,all of the immunoglobulin light chains genes belong to 3 different isotypes were obviously up-regulated,although reached their peak levels at different time. Only LycIgL3 was slightly down-regulated after stimulation with bacterial vaccine in spleen, and then recover to nomal and up-regulation in 72h.All of these results indicating that it occurred at the early phase of induction and was differentially modulated in the two tissues by different stimulation.The up-regulation of LycIgL by bacterial vaccine or poly(I:C),a virus mimic,might also be the result of NF-kB activation induced by bacterial vaccine or poly(I:C).We also simulate the homology modeling of LycIgL of large yellow croaker.All of the three IgL isotypes contain two domains,the same with the IgL in human,which is useful for the further study of the relationship between structure and function.