Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8498340 | Fish & Shellfish Immunology | 2018 | 40 Pages |
Abstract
Invertebrates, unlike vertebrates which have adaptive immune system, rely heavily on the innate immune system for the defense against pathogenic bacteria. Lysozymes, along with other immune effectors, are regarded as an important group in this defense. An invertebrate-type (i-type) lysozyme, designated Urechis unicinctus invertebrate-type lysozyme, Uu-ilys, has been isolated from nephridia of Urechis unicinctus using a series of high performance liquid chromatography (HPLC), and ultrasensitive radial diffusion assay (URDA) as a bioassay system. Analyses of the primary structure and cDNA cloning revealed that Uu-ilys was approximately 14â¯kDa and composed of 122 amino acids (AAs) of which the precursor had a total of 160 AAs containing a signal peptide of 18 AAs and a pro-sequence of 20 AAs encoded by the nucleotide sequence of 714 bp that comprises a 5â² untranslated region (UTR) of 42 bp, an open reading frame (ORF) of 483 bp, and a 3â² UTR of 189 bp. Multiple sequence alignment showed Uu-ilys has high homology to i-type lysozymes from several annelids. Relatively high transcriptional expression levels of Uu-ilys was detected in nephridia, anal vesicle, and intestine. The native Uu-ilys exhibited comparable lysozyme enzymatic and antibacterial activities to hen egg white lysozyme. Collectively, these data suggest that Uu-ilys, the isolated antibacterial protein, plays a role in the immune defense mechanism of U. unicinctus. Recombinant Uu-ilys (rUu-ilys) produced in a bacterial expression system showed significantly decreased lysozyme lytic activity from that of the native while its potency on radial diffusion assay detecting antibacterial activity was retained, which may indicate the non-enzymatic antibacterial capacity of Uu-ilys.
Keywords
TFARapid amplification cDNA endsHEWLUrechis unicinctusTSBNAGQ-TOFUPLCRT-qPCRIPTGORFMECPBSCFUN-acetylmuramic acidamino acidTrifluoroacetic acidsodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGEisopropyl β-D-1-thiogalactopyranosidehen egg white lysozymeRecombinant protein productionProtein isolationreal-time quantitative PCRquadrupole time-of-flightTryptic soy brothopen reading framePhosphate-buffered salineRaceUTR یا untranslated regions untranslated regionNAMN-acetylglucosaminepolymerase chain reactionPCRhigh performance liquid chromatographyHPLCcolony forming unit
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Authors
Hye Young Oh, Chan-Hee Kim, Hye-Jin Go, Nam Gyu Park,