Molecular Cloning And Immunological Characterization Of Akirin In Common Carp (Cyprinus Carpio L.)

The innate immune system is the first line of all metazoans defense against invading microorganisms. Akirin, a new nuclear factor was discovered in the innate immune response of Drosophila melanogaster, is a small molecule nuclear protein composed of about 200 amino acid residues, play a critical role in the immune deficiency (Imd) pathway. Akirin proto-orthologue gene highly exists in invertebrate metazoans to higher vertebrates and the sequences are highly conserved. In Imd pathway of drosophila and Toll pathway of vertebrate, Akirin affects the transcription of genes regulated by the transcription factor known as NF-κB. The animals' susceptibility to infection with Gram-negative bacteria will be increased when reducing Akirin levels using RNA interference.In this paper, we have isolated and sequenced the full-length cDNA of Akirin1 and Akirin2 from the liver of common carp by the method of molecular cloning and rapid-amplification of cDNA ends (RACE). The primer pairs used for experience are designed on the basis of the conserved region of zebrafish compared with other animals. As well as studied their amino acid sequence alignment and phylogenetic analysis between carp Akirins and insect and vertebrate Akirins. Then we studied the expression pattern of Akirin1 and Akirin2 in tissues in relatively health common carp by RT-PCR. The tissues include the liver, spleen, foregut, hindgut, gill, head kidney, blood, muscle, skin and oral epithelium. Studies have shown that Akirin1 can regulate muscle regeneration and cell chemotaxis, while Akirin2 play as a transcription factor joined the innate immunity. So we choose Akirin2 for the following studies. Akirin2 gene exprssion level were detected by real-time quantitative PCR in the liver, spleen, head kidney, foregut and hindgut of common carps which were stimulated by Vibrio anguillarum, through which can verifies the function of the carp Akirin in the innate immunity system.The full length Akirin1 cDNA exhibited 1204bp and contains a 179bp 5'-untranslated region (UTR), a 570bp open reading frame (ORF) and a 455bp 3'-UTR. The open reading frame (ORF) encoding a protein of 189 amino acids, and there are a nuclear localization signal (NLS) sequence KRRRC between position 23 and 27. The full length Akirin2 cDNA exhibited 1290bp and contains a 192bp 5'-UTR, a 555bp ORF and a 543bp 3'-UTR. The ORF of Akirin2 encoding a protein of 184 amino acids, and there are also contains the NLS sequence KRRRC between position 23 and 27.The sequence and phylogenetic anlysis of carp Akirins show that they are highly similar about 57.2%. A sequence alignment of carp Akirins and some other known and predicted Akirins are performed at the amino acid level. The deduced amino acid sequence from carp Akirin1 and Akirin2 cDNA was highly similar to Akirins of zebrafish (Akirin1 97.9%, Akirin2 89.4%), Rainbow trout (Akirin1 87.0%, Akirin2 79.9%) and salmon (Akirin1 86.5%, Akirin2 79.9%). To determine the phylogenetic relations between insects and vertebrates Akirins, a phylogentic tree was constructed with the amino acid sequence of these akirins. The insects's Akirins are in different branches with vertebrates, and in the branch of vertebrates, Akirin1 and Akirin2 are in two parallel branches. From this, Akirin1 and Akirin2 existence evolutionary conservation, suggesting that there may be differences between the two functions.RT-PCR demonstrated that, in common carp, Akirin1 mRNA transcripts were highly abundant in liver, spleen, foregut, hindgut, gill, head kidney, muscle, skin and oral epithelium, less abundant in whole blood. Akirin2 mRNA transcripts were highly abundant in liver, spleen, foregut and head kidney, abundant in gill, muscle and skin, less abundant in hindgut and whole blood.Expression of Akirin2 were measured in previously non-exposed fish, 6 hours, 1, 2, 3 and 5 days after experimental Vibrio anguillarum challenge to determine if it is associated with host response to V. anguillarum infection by real-time quantitative PCR. The mRNA copy numbers of carp Akirin2 were increased after 6 hours in some organs and have reached the maximum, which expressed in the liver amounted to about 2 times then the normal expression level, and the expression in other organs are more then 1 times. The expression level was decreased after 1 day.Carp is an important freshwater fish in our country, and study the innate immune system of carp will to improve the theoretical basis for the fish innate immunity and make more economic values of carp cultivation. This paper clone carp Akirin1 and Akirin2 full-length cDNA, and studied their amino acid sequence alignment and phylogenetic analysis, then research carp Akirins gene expression and immune functions.