| Nitric oxide is a multifunctional gaseous biological messenger, produced by a variety of cells or tissues, it can reduce or even kill the infection factor, and excessive nitric oxide may cause tissue injury and accelerate the process of inflammation inducible nitric oxide synthase(i NOS) is a key enzyme of nitric oxide molecules, thus grasp the mechanism of i NOS it is crucial to its regulation. The experiment aims to obtain the full-length c DNA sequence of i NOS, for the further research of the regulation mechanism of i NOS protein in common carp that using of RT-PCR, Northernblot and other methods, and detected by i NOS fluorescence quantitative expression in vitro and in vivo after stimulation, and whether the expression has tissue specificity.Objective: to obtain the carp inducible nitric oxide synthase c DNA sequence,and in vivo and in vitro on expression of i NOS in stimulating factor stimulated and tissue specific expression; experimental methods: the sequence of EST c DNA Library of peripheral blood white blood cells to carp in the i NOS based on the library screening and c DNA rapid amplification of 5 ’rapid amplification of c DNA ends(5’-RACE), obtained the full c DNA sequence of i NOS in carp, in vitro experiment, with LPS(1.0 μg/m L) stimulation of peripheral blood leukocytes in primary cultured raised 4 h, 12 h, concanavalin A(Con A, 1.0 μg/m L)stimulation 4h, 24 h, extraction of total RNA from peripheral blood leukocytes; in vivo respectively by Aeromonas hydrophila natural flagellin, recombinant protein fla A and fla B stimulated carp 4 h, 12 h, extraction, head kidney total RNA; and by Aeromonas hydrophila 4 h after the extraction of infected carp liver, spleen, heart,kidney, muscle, gill and brain RNA; using RT-PCR method for detection of i NOS in different tissues(cells) were analyzed and the expression of m RNA on the level of i NOS. Results: the full-length c DNA fragment of a total of 3 704 bp, contains 745’ non encoding region, 246 bp 3’ non encoding region, a 3 384 bp complete open reading frame(ORF), encoding 1 127 amino acids. Sequence homology with the sequence of i NOS gene showed that carp 94% homology; RT-PCR results showed that by LPS the amount of i NOS, increased expression of Con A stimulated 4H of white blood cells, with prolonged stimulation(12 h, 24 h) expression decreased,with the passage of time does not continue to increase; the natural flagellin and recombinant protein fla A, fla B stimulated at 4 h and 12 h, the amount of the same trend of the expression of i NOS, expression the i NOS groups in head kidney Volume increased, but 4 h increased more obvious; i NOS was detected in all tissues,including in the head kidney expression was the highest, followed by the gill was found with Aeromonas hydrophila infection in carp, brain, spleen, liver, in heart and muscle expression was the lowest. Study on the regulation of the expression of i NOS, will provide experimental materials for further proven fish immune response mechanism to foreign pathogens stimulate, also, can lay an important foundation for a more thorough understanding of the mechanisms of innate immunity in fish. |
