Construction Of SMART CDNA Library From Bacteria-induced Kidney And Function Analysis Of PA28-β And CD59 Gene From Large Yellow Croaker

Large yellow croaker (Pseudosciana crocea) is a proper economic fishes in China. It is welcomed because of its good taste and medical values. Artificial aquaculture of large yellow croaker was developed enormously after breeding and acquaculture technology was handled. Bacterial and viral diseases, which brong huge damage for acquaculture, were induced by large scale acquaculture and worsing water condition. The innate immunity of fish has a fast response for various pathogeny. It was studied extensively because its important value in fish immune system.In order to study the expression pattern and action mode of immune-relevant genes, inactivated trivalent vaccine consisting of Vibrio alginolyticus,V. paraphamolyticus, and Aeromonas hydrophila was employed to stimulate the immune system of large yellow croaker.Kidneys and blood were sampled at 6,12,30,36 h post-induction.Anti-bacteria activity,bacteria lysis activity and hydroxybenzene oxydase activity were ditected in use of Serum derived from bood samples.Results demonstrated that the sample at 30 h post-induction could be the best to construct the SMART cDNA library.Then,mRNA was isolated and cDNA was synthesized. The SMART cDNA library was constructed ,when the ligation product of cDNA and pcDNA3.0 was transformed into E.coli JM109 competent cells.Colony PCR results showed that the recombinant rat and library capacity was up to 97.7 % and 1.4×10~5 clones respectively.The SMART cDNA of kidney is the base of EST(Expressed sequence tag) analysis.Antigenic peptides presented on MHC class I molecules to cytotoxic T cells are generated in the cytosol by the 20S proteasome. Two activators PA28-α and PA28-β, which are inducible by interferon-γ (IFN-γ), activate the latent 20S proteasome, thus playing an important role in the processing of MHC class I antigen. Molecular properties and function in the MHC class I antigen processing of PA28 have been well studied and documented in mammals while little is known in fish. In the present study, we reported the cloning of a PA28-β gene homologue from the spleen of large yellow croaker (Pseudosciana crocea), an economically important marine fish (LycPA28-β). The full length cDNA of LycPA28-β is 1133 nucleotides (nt) encoding a protein of 245 amino acids (aa), with a putative molecular weight of 27.7 kDa. The deduced protein shares 76, 69, 61, 60, 59, 57 and 57% sequence...