| Octopus ocellatus is an important economic specie in coastal areas. Because of a gradual worsening of situation in aquaculture and marine ecology, understanding the immunologic mechanism of O. ocellatus on molecular level was an urgent need. It could improve germplasm quality,resistance and provide theoretical support for the sustainable development of marine ecology and aquaculture.Due to the lack of acquired immunity, O. ocellatus mainly dependents on innate immune system to resist the invasion of microorganisms.Lysozyme which can induce the synthesis and secretion of other immune factors is acrucial immune effector in response of immune defense. Lysozyme has an enzymatic activity to cleave the β-1,4-glucosidic bonds linkage between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan in bacterial cell walls. According to the source and amino acid sequence, lysozymes are classified into six types including chicken type, goose type, plant type, bacterial type, phage type, and invertebrate type. Among these types, i-type lysozymes were only be discoved in invertebrates and their reported mainly focused on Annelida, Echinodermata, Mollusca and Arthropoda.In this study, the full-length c DNA encoding Oo Lys was indentified from the c DNA Library of O. ocellatus. Bioinformatics of Oo Lys were analyzed by various biological Software. The m RNA expression patterns of Oo Lys, both in tissues and in haemocytes after Listonella anguillarum and Micrococcus luteus challenge were then characterized by Real-time PCR(RT-PCR), respectively. Recombinant lysozyme protein(r Oo Lys) in vitro was express in BL21(DE3) for expression of prokaryotic expression vector system.Molecular biology and immunobiology methods were used to analyze enzymatic activity of r Oo Lys, aiming at exploring the role it plays in the immune defense mechanism in O. ocellatus, and providing theoretical support for the development of marine shellfish immunology.The full-length c DNA of Oo Lys was 678 bp. The complete sequence of Oo Lys c DNA consisted of a 5′ terminal untranslated region(UTR) of 156 bp, a 3′ UTR of 111 bp with a poly(A) tail and a polyadenylation signal AATAAA, and an open reading frame(ORF) of 411 bp. The ORF encoded a polypeptide of 137 amino acids with an isoelectric point of 8.15 and predicted molecular weight of 15.36 k Da. SMART analysis showed that the amino acids 1(M)~18(A) encoded a signal peptide, while 19(A)— 135(K) constituted a Destabilase domain. Analysis of sequence homology and phylogenetic trees revealed that Oo Lys had a closed relationship with other bivalves in Mollusca such as Ruditapes philippinarum, Chlamys islandica and Meretrix meretrix. Three conservative sites in activity center closely related to the enzymatic activity were Glu(40), Glu(49) and Ser(52) were. The analysis of three-dimensional structure showed that 12 conserved cysteine residues were involved in the formation of four pairs of disulfide bonds, and they might enhance the stability of protein conformation. These results indicated that Oo Lys were i-type lysozyme.Quantitative Real-time PCR(RT-PCR) was used to determine m RNA expression of Oo Lys in various tissues. The results showed that Oo Lys was widely expressed in a range of tissues including hemocyte, muscle, gill, hepatopancreas, saccus renalis, mantle, stomach, branchial heart, systemic heart, and gonad. The Oo Lys m RNA was expressed with the highest level in hepatopancreas which was 310-fold compared with that in gonad, while the lowest in gonad. The RT-PCR analysis was also employed to study the m RNA expression of Oo Lys m RNA in hemocytes after the O. ocellatus were stimulated by Listonella anguillarum and Micrococcus luteus. The m RNA expression of Oo Lys showed a obviously induced trend, and it was first significantly up-regulated to 7.3 and 4.3-fold of blank group at 6 h post Listonella anguillarum and Micrococcus luteus, respectively. The first up-regulation implied that Oo Lys might be involved in the acute stress response. The m RNA expression reached peak to 9.7 –fold of blank group at 48 h post Listonella anguillarum stimulation, while the second significantly up-regulation occured at 24 h post Micrococcus luteus stimulation. During this period Oo Lys might be involved in the removal process against pathogenic microorganisms.The Oo Lys domain was cloned into p EASY-E1 expression vector. The recombinant expression plasmid was transformed into competent Escherichia coli BL21(DE3), expressed with 1 ‰ IPTG at 37 oC. The recombinant Oo Lys(r Oo Lys) with His-Tag label was purified by FF affinity chromatography after ultrasonic disintegration, and then renaturated by a dialysis against decreasing linear gradient of urea solution.Lytic activity andmuramidase activity of r Oo Lys was measured using turbidimetric assay. Two gram-positive bacterium, M. luteus, S.aureus, and two gram-negative bacteria, E. coli, L. anguillarumwere chosen for the test of lytic activity. The r Oo Lys had lytic activity against gram-positive and gram-negative bacteria, while the activity against gram-positive bacteria was stronger. The antimicrobial effect on gram-negative bacteria and gram-positive bacteria was inhibited by antisera against r Oo Lys. The heat treatment could also inhibit the antibacterial activity of r Oo Lys against gram-positive bacteria, but could not effect its activity agaist gram-negative bacteria. It revealed that antibacterial activity against gram-negative bacteria could be achieved through enzymatic or non-enzymatic pathway, while its antibacterial activity against gram-positive bacteria depended on enzymatic ways. Peptidoglycan which was the main ingredient of positive bacteria cell wall was chosen as reaction substrate to measure muramidase activity of r Oo Lys. The absorbance value decreased significantly indicated r Oo Lys could degrade peptidoglycan. Its antibacterial activity might depend on muramidase activity by hydrolyzing the β-1,4- glycosidic bond cleavage bacteria.The inhibitory effect of r Oo Lys on L. anguillarum was detected by urbidimetric assay and SEM assay. Absorbance value of r Oo Lys experimental group was significantly lower than PBS group and Amp group after 4 h incubation. In SEM assay, a serious deformation was observed in L. anguillarumin SEM assay. The surface of L. anguillarum become sroughness and the cytoderm was damaged. It revealed that r Oo Lys had distinct inhibitory effect on L. anguillarum. These results applied theoretical supports to actual production of aquaculture.In summary, an i-type lysozyme gene(Oo Lys) from O. ocellatus was cloned and analyzed, then its expression pattern was tested and its function was studied. It confirmed that Oo Lys participated in immune response against gram-negative and gram-positive bacteria and its antibacterial activity against gram-negative bacteria could be achieved through enzymatic or non-enzymatic pathways. The r Oo Lys has muramidase activity and it could partly damage L. anguillarum. All these results revealed that Oo Lys played an important role in the immune defense of O. ocellatus. This study shed new light on further understand O. ocellatus immune mechanisms and provided was to, and provide academic references to resistance improvement. |
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