Molecular Cloning, Charaterization And Function Of Immune-related Genes From Scophthalmus Maximus And Paralichthys Olivaceus

Turbot and flounder are important economically marine fish. Recently, the deterioration of mariculture environment cause the frequency of fish diseases, which results in increasing and appreciable economic damage, and also has adverse effect on the healthy development of aquaculture. Non-specific immunity system plays an important role in the defense of external stimuli and pathogen invasion in fish. Therefore, analyzing the function of immune-related gene of fish and clarifing the network regulation process by using molecular biology technology and genetic engineering are of great value for the development of disease-resistant fish and medical research, in terms of both theoretical and social significance.When resisting to pathogen, secretion of antimicrobial peptides (AMPs) was one of naturally immune response in fish. Hepcidin is a small molecule peptide and one of the imprortant members of these AMPs, which also plays a significant role in regulating the concentration of intracellular iron.Toll-like receptor (Toll-like receptor, TLR) family is the major receptor when the host cell identifies the various components of pathogenic microorganisms, and NF-κB is located in the hub of the TLR signaling pathways downstream. When cells are activated by biological stress stimulation, activated NF-κB goes into the nucleus to regulate the expression of inflammatory cytokines, and triggers the innate immune and the acquired immunity for resistance to pathogenic microorganisms. Akirin is newly identified to be a member participating in the NF-κB pathway.To clarify the function and the network-regulation mechanism of immune-related genes in turbot and flounder, we analyze the charaterization and function of several immune-related genes, such as SmHepcidin and SmAkirin from turbot, PoAkirin from flounder.First, the full length of SmHepcidin mRNA was obtained by using the RACE method, and its promoter region was also cloned by using genome walking method. The motif sequence analysis of promoter of SmHepcidin showed that the region contains the binding sites for transcription factor NF-κB, and SmHepcidin may have a feedback regulation for NF-κB through dual-luciferase assay. In addition, the fact that exogenous ScHepcidin proteins can regulate cell iron content has further verified the function of Hepcidin. Moreover, the full-length cDNA sequence of the regulation-related factors of SmHepcidin - SmFPN1 (Ferroportin 1) and SmTFR2 (Transferrin Receptor) were cloned. After treated by bacteria or virus, or injected by exogenous ScHepcidin protein, expression changes of SmHepcidin, SmFPN1 and SmTFR2 in the liver, spleen and kidney tissues were detected by real-time quantitative PCR method. The results showed that, with the increasing expression of SmHepcidin, the expression level of SmFPN1 was significantly reduced, while the expression level of SmTFR2 was decreased or has no notable change. The RNAi research of the expression patterns of these three genes in turbot kidney cells also confirmed the above results: when the expression level of SmHepcidin decreases, the expression level of SmFPN1 was significantly up-regulated and the expression level of SmTFR2 was also reduced a little. In summary, it is hypothesized that SmHepcidin, SmFPN1 and SmTFR2 play an important role in the regulation of iron homeostasis, the expression level of SmHepcidin rapidly increases when pathogen invades, and regulate other related genes expression to change the iron concentration in cells in order to resist pathogen invasion. All these findings indicate that SmHepcidin play a defensive role in resisting the invasion of pathogens.Second, the full-length cDNA of SmAkirin1 was obtained by using method of RACE, and protein prediction shows that SmAkirin1 was a nuclear protein. By using SmAkirin1-GFP and SmAkirin1-m-GFP fusion protein plasmid to transfect turbot kidney cell line, the results confirmed the conclusion that nuclear localization signal (NLS) presents in the N terminal of SmAkirin1. Although the protein localized in the nucleus and participated in the regulation of immune response genes, SmAkirin1 is not a transcription factor due to the fact that SmAkirin1 has no self-activation activity. After bacteria or virus treatment, the expression of SmAkirin1 in the liver, spleen, kidney tissues, and SMKC was detected by using real-time quantitative PCR method. The results showed that SmAkirin can be induced by bacteria and viruses.In the analysis of immune-related genes in flounder, a yeast two-hybrid cDNA library of flounder was constructed. The titer of this original library was 1.6×10⁶ cfu/mL, The titer of the primary library glycerol is about 4.1×10⁴ cfu/1.8mL, and the capacity of the amplification library was approximately 5.0×10¹⁰ cfu / mL, and capacity after transformed into yeast cells is about 5.4×10⁶ cfu/mL. We found a variety of immune-related genes from this library, and cloned the full-length cDNA sequence of PoAkirin1. By using PoAkirin1 as the bait to screen the interacting proteins, a total of 49 positive clones were screened, and 7 possible interacting genes were acquired. Two possible immune-related proteins or proteins located in the nucleus through protein prediction were selected, and tested by yeast rotary verification and segment deletion verification, the result showed that these two proteins (PoC1q and PoHEPN) can be combined with PoAkirin, and interacting sites between PoHEPN and PoAkirin may be located at the N terminus. After bacteria or virus treatments, relative expression changes of PoAkirin, PoC1q and PoHEPN are detected in the liver, spleen and kidney tissues by using real-time quantitative PCR. The results showed that all these three genes can be induced by pathogens, and may locate in the different positions of signal transduction pathway according to their different response time. Furthermore, the recombinant proteins of PoAkirin and PoHEPN were constructed and expressed in E. coli, which provides the foundation for identification of interaction between PoAkirin and PoHEPN and further research on their charaterization and function.