| Grass carp(Ctenopharyngodon idella)is an important economic freshwater fish in China,accounting for a high proportion of total freshwater fishery production.However,the frequent occurrence of various diseases caused by pathogens in the water has a certain impact on the output value.Innate immunity is an important line of defense for the fish immune system.It recognizes pathogen structures through pattern recognition receptors(PRRs)and induces immune responses in the body.Therefore,it is necessary to study the innate immune gene of grass carp,and provide new ideas for breeding new grass carp varieties with strong disease resistance.Growth arrest and DNA damage inducible 45-beta(Gadd45B)is essential for mitogen-activated protein kinases(MAPK)activities,and involved in regulating growth,apoptosis,and DNA demethylation.In this study,the full-length cDNAs of gcGadd45 Ba and gcGadd45 Bb genes in grass carp were cloned,and sequences were analyzed and grass carp and cells were treated with Aeromonas hydrophila as pathogen models.The expression of grass carp individuals and cells after bacterial infection,and design experiments through the construction of overexpression vectors and recombinant proteins to explore the function of the gene,the following results were obtained:1.A short fragment of gcGadd45 Ba and gcGadd45 Bb cDNA was obtained from the grass carp transcript,and the accuracy of the primer was verified.The full-length sequence was obtained by RACE.The full-length cDNA of gcGadd45 Ba was 1279 bp,including the 650 bp 5' untranslated region(UTR),a 149 bp 3'-UTR,and a 480 bp ORF encoding a 159 amino acid protein with a molecular weight of 17.91 kDa and a theoretical isoelectric point of 4.38.The full-length cDNA of gcGadd45 Bb is 1190 bp,including a 564 bp 5'-UTR,140 bp.The 3'-UTR and 486 bp ORFs encode a protein of 161 amino acids with a molecular weight of 17.97 kDa and a theoretical isoelectric point of 4.51.Analysis of its amino acid sequence revealed that the protein encoded by gcGadd45 Ba does not have a hydrophobic region,and the protein encoded by gcGadd45 Bb has a weak hydrophobic region,which provides us with the possibility to synthesize soluble recombinant proteins.2.The normal tissue fluorescence quantitative PCR results of gcGadd45 Ba and gcGadd45 Bb showed that they were expressed in all tested tissues of grass carp.And the gcGadd45 Ba gene is relatively high in liver,heart,muscle,skin,blood and spleen.The gcGadd45 Bb gene is expressed relatively high in blood,fins,heart and spleen.The levels of gcGadd45 Ba and gcGadd45 Bb showed significant differences in all tested tissues except for fins and ticks.The expression of immune tissues showed that gcGadd45 Ba and gcGadd45 Bb had significant immune responses after Aeromonas hydrophila infection,and the expression level between 4 h and 12 h showed an upward trend,gradually decreased between 12 h and 72 h,and existed.gcGadd45 Ba is highly expressed in the spleen and gcGadd45 Bb is highly expressed in the head kidney.3.By constructing the overexpression vector of gcGadd45 Ba and gcGadd45 Bb,the transcription of downstream related immune factors(IL-8,IL-1?,TNF-?)was detected after transfection into CIK cells,and the results showed that the transcription of the downstream immune factor could be significantly activated.Moreover,it can be seen in the bacteriostatic experiment that the CIK cells overexpressing the gene of interest can significantly inhibit the efficiency of bacterial invasion.4.The recombinant protein of gcGadd45 Ba and gcGadd45 Bb was expressed and purified in vitro,and the experiment of designing blood cell phagocytic bacteria was carried out.The bacteria were carried by FITC fluorescent labeling,and the fluorescence signal was detected by flow cytometry to measure the effect of recombinant protein on the efficiency of blood phagocytic bacteria.The results showed that both gcGadd45 Ba and gcGa-dd45 Bb have serum-dependent hematopoietic phagocytosis. |
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