Cloning And Expression Of Three Immune Genes In Sea Cucumber, Apostichopus Japonicus And Their Involvement In Immunity

Sea cucumber(Apostichopus japonicus) is an economically important farmed echinoderm species in the North of China. Lack of information on the innate immune system of sea cucumber has prevent our research on their nutrition and immune defense mechanisms. NF-κBs were the key factors in animal innate immunity system, which multiplates the expression of a lot of immune factors such as cytokines. Lysozyme plays an important role in disease resistant in marine animal. The cDNAs of Aj-rel, Aj-p105and Aj-lysozyme were cloned and expressed in the studies. The characteristics and functions of the three genes could help us to get further information of the inmmune defence mechanisms in A japonicus. The effective expression of Aj-lysozyme could also be exploited as immunostimulant. The main research contents and results were stated as follows:(1) cDNA products of Aj-Rel and Aj-p105were amplificated using RT-PCR technology. The full-length cDNA sequences of Aj-Rel and Aj-p105were obtained based on3'RACE and5'RACE approach. Aj-rel and Aj-p105share numerous signature motifs with their vertebrate orthologues, notably the Rel homology domain, Rel protein signature DNA binding motif, nuclear localization signal, and the ankyrin repeats and DEATH domain for Aj-p105. Phylogenetic analyses indicate that Aj-rel and Aj-p105belong to the Rel-like and NF-κB-like protein respectively. Based on the sea cucumber lysozyme sequence in Genebank, DNA fragment coding for the mature peptide of A. japonicus lysozyme was amplified using RT-PCR technology and was confirmed by DNA sequencing.(2) The selected region of Aj-Rel and Aj-p105were expressed in E. coli. The recombinant fusion proteins were purified using HisTrap FF column. New Zealand White rabbit were immunized with the purified proteins and antiserum were prepared. Western Blot analysis indicate that the antiserum prepared can be used to detect Aj-rel, Aj-p105and Aj-p50. Co-immunoprecipitation followed by Western Blot was used to study the interactions of Aj-p105with Aj-rel, Aj-p105and Aj-rel were immunoprecipitated and their binding partners were detected by Western Blot. Immunoprecipitation of Aj-Rel resulted in the co-precipitation of Aj-p105and Aj-p50indicated that Aj-Rel can form heterodimers with Aj-p105and Aj-p50. Immunoprecipitation of Aj-p105and Aj-p50resulted in the co-precipitation of Aj-Rel, which further proved their interaction.(3) Aj-lysozyme gene was cloned into the expression vector pPIC9K. pPIC9K-lys was transformed into P. pastoris GS115(his4) and the highest expression strain of pPIC9K-lys from His+Muts phenotype strains were selected and then used to be expressed in Pichia pastoris. The expression condition was optimized and the highest expression level was achieved under the condition of1%methyl alcohol and pH5after192h, the optimized expression level can reach50μg/ml. The antiserum was prepared using the same method. Aj-lysozyme exhibited stronger antimicrobial activity than HEW lysozyme toward Micrococcus lysodeikticus and Vibrio splendidus under the same condition, as detected by growth inhibition on agar plate and turbidity assay.(4) The mRNA expression pattern of Aj-rel, Aj-p105and Aj-lysozyme following LPS infection was analyzed using Real-time PCR approach, and their protein expression pattern was analyzed using Western Blot approach. Results indicate that LPS can improve the immunity of A. japonicus through the NF-κB pathway. The factors in the LPS-inducted NF-κB pathway of A. japonicus is fast acting. It is a conserved signal transduction pathway that is functionally compatible with the canonical pathway in mammalians suggesting that the roles of NF-κB have coevolved and remained conserved from the sea cucumber to humans, playing an archaic but crucial role in the innate immune response.In this paper, the cDNAs of Aj-rel, Aj-p105and Aj-lysozyme were cloned and expressed in E. coli and P. pastoris expression vector respectively and the antiserum were prepared. Co-immunoprecipitation proved the interaction of Aj-p105with Aj-rel, Aj-lysozyme exhibited stronger antimicrobial activity toward Micrococcus lysodeikticus and Vibrio splendidus than HEW lysozyme. This results will help us understand the immune system and their function in A japonicus. The mRNA and protein expression pattern of Aj-rel, Aj-p105and Aj-lysozyme following LPS infection increased the available information on the defense mechanisms and regulation processes of NF-κB-pathway which are of pressing practical relevance in disease control and drug exploitation.