Cloning And Immunological Function Of IRF8-like Gene In Chlamys Farreri

The family of Interferon regulatory factors(IRFs)is a class of transcriptional regulators with important biological functions in host innate immune defense and stress response.Currently,vertebrate IRFs and their mediated interferon antiviral immune pathways have been studied relatively thoroughly;however,further studies are needed to determine the functions of IRFs in invertebrates.In this study,an IRF gene(named Cf IRF8-like)was cloned from the Zhikong scallop Chlamys farreri using molecular biology,cell biology and bioinformatics,and its natural immune function was initially investigated.The main results and discussions are as follows:1.The complete coding sequence of an IRF gene was obtained from the Zhikong scallop(C.farreri)and named Cf IRF8-like.the open reading frame of Cf IRF8-like is1371 bp long and encodes 456 amino acids.Protein structure prediction showed a typical IRF structural domain at the N-terminal end and a typical IRF3 domain at the C-terminal end of the Cf IRF8-like protein.Multiple sequence comparisons confirmed the conserved amino acid sequences of the functional structural domains of these two proteins.Phylogenetic analysis showed that the identified shellfish IRF proteins clustered into two branches(IRF8 subclass and IRF1/2 subclass),and Cf IRF8-like belonged to the IRF8 subfamily of shellfish proteins.Since both contain the typical IRF3 structural domain at the C-terminus,the shellfish IRF8 subfamily clusters with the vertebrate IRF3,IRF4 and IRF5 subfamily proteins.2.Fluorescence quantitative PCR(qRT-PCR)results showed that the expression of Cf IRF8-like m RNA was detected in all tested scallop tissues,and the highest expression was in gill tissues.Meanwhile,the expression of Cf IRF8-like transcriptome in gill tissues could be significantly induced by poly(I:C)stimulation.3.The results of protein interaction experiments showed that Cf IRF8-like protein could directly bind to TBK1/IKKε family protein(Cf IKK2)of scallop through its N-terminal IRF structural domain.It revealed that an ancient functional TBK1/IKKε-IRF signaling axis may exist in scallops.4.The results of dual luciferase reporter gene experiments showed that overexpression of Cf IRF8-like in human embryonic kidney 293 T cells specifically activated the mammalian interferon β promoter and interferon-stimulated response element promoter in a dose-dependent manner,tentatively confirming the critical role of Cf IRF8-like in activating the mammalian interferon pathway.In summary,this gene may be involved in the natural immune process of host cells,and the preliminary results of signal transduction and immune function of Cf IRF8-like protein may lay the foundation for an in-depth understanding of the innate immune function of invertebrates,while the experimental results also provide theoretical support for the breeding of disease-resistant strains of scallops.