Preliminary Study Of The Function Of IKKε Gene Of Polyploid Fish

The innate immunity is also known as non-specific immunity, referring to the immune ability which is naturally owned by individuals and have a wide range of effects, as well as not for a specific antigen. Innate immunity is described as a kind of immune action which contains numerous pattern recognition receptors (PRR) and is highly complicated and rapidly induced. Those pattern recognition receptors(PRR) can recognize different pathogen-associated molecular patterns (PAMPs) and activate immune responses of organisms by different signal pathways.IKKs(NF-kB kinase ε) is a kind of kinase of Toll-Like and NF-kB signal pathways in innate immune system. It is a newly found member of IkB kinase family and was firstly identified as the serine/threonine kinase induced by lipopolysaccharide. IKKε phosphorylates IkBα through signaling molecules to induce the activation of NF-kB or phosphorylates IRF3and IRF7kinases with TBK1under the action of caffolding protein, thus inducing secretion of IFN.The innate immunity is the foundation for fish and other lower vertebrates to defend the invasion of disease. Lots of gene elements which exist in the innate immune signal pathways of mammals are also conservative in teleosts. At present, TLR,RIG-I and MAVS genes have already become a hotspot for studying innate immunity in fish. However, datas about the functions of IKKε in fish in innate immunity were seldom shown in reports.In this paper, we cloned the whole sequence of IKKε cDNA of grass carp and black carp which are important commercial fishes of Cyprinidae family. We also cloned a part of cDNA sequence of IKKε genes of AT/RCC/TCC and initially study the functions of IKKε genes in black carp. The contents and results of this paper are shown as below:1. The full length of IKKε cDNA of black carp is2537bp,among which the5’ noncoding region contains285base pairs. The full length of open reading frame(ORF) is2169bp and its3’noncoding region contains83base pairs. It is predicted that they can encode723animo acids and the molecular weight of the protein is82556. The full length of IKKε cDNA of grass carp is2529bp,among which the5’noncoding region contains274base pairs. The full length of open reading frame(ORF) is2169bp and its3’noncoding region contains86base pairs. It is predicted that they can encode723animo acids and the molecular weight of the protein is82476.9.2. By comparing the homology of IKKs genes between black carp and grass carp, we can conclude that the homology between them are very high,reaching97.3%; But the sequence homology betwwen them and zebra fish are73.4%; The sequence homology betwwen them and mammals/birds are relatively low. By analysing phylogenetic trees, we can find that the IKKs genes of black carp and grass carp belong to the same branch,which are consistent with their high suquence homology. After the analysis of the amino acid sequences of IKKs in black carp and grass carp, we discovered that both of their protein sequences contain the protein domain that are similar with IkB kinase family.3. According to the sequence of pcDNA5-FRT/TO vector, lentivirusLinker vector, we constructed the recombinant plasmid of IKKs which has been sequenced and the vectors of pcDNA5-FRT/TO-FLAG-BC-ikk, pcDNA5-FRT/TO-BC-ikk-FLAG,CDH-CMV-Puro-FLAG-BC-ikk and CDH-CMV-Puro-BOikk-FLAG. HEK293T cells were transfected with the recombinant plasmid by calcium phosphate transfection method. Through Westen blot, we have identified and confirmed that IKKe genes of black carp can be well expressed in HEK293T cells and molecular weight of the protein are about85KD.4. NIH3T3cells and EPC cells were transfected with recombinant plasmid FRT/TO-Flag-bcIKKε and FRT/TO-bcIKKε-Flag through Lipofectamine2000. The results of confocal and immunofluorescence showed that IKKe in black carp are mainly expressed in cytoplasm.5. We cloned the part of the open reading frame of ATYRCC and part of cDNA for TCC, with85bp for its3’-UTR.In summary, in this study we have cloned the whole sequence of IKKε gene of Black carp and Grass carp,and part of the sequence of IKKε gene of AT/RCC/TCC. Besides, we have made preliminary research on IKKε gene expression in black carp and intracellular location.This paper has laid a solid foundation for systematic functional study of bcIKKe,gcIKKε and the IKKε gene of AT/RCC/TCC in the innate immune.