Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides
Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-...
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| Veröffentlicht in: | Fish & shellfish immunology 2012-08, Vol.33 (2), p.186-196 |
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| creator | Wei, Shina Huang, Youhua Cai, Jia Huang, Xiaohong Fu, Jing Qin, Qiwei |
| description | Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and “GSTDYGIFQINS” motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.
► c-type lysozyme gene from Epinephelus coioides (Ec-lysC) was identified and characterized. ► The expression of Ec-lysC in response to LPS, SGIV and Vibrio alginolyticus were different. ► The recombinant Ec-lysC had lytic activities against Gram-positive bacteria and Gram-negative bacteria. ► Ec-lysC overexpression decreased SGIV replication in grouper cells. |
| doi_str_mv | 10.1016/j.fsi.2012.03.027 |
| format | Article |
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► c-type lysozyme gene from Epinephelus coioides (Ec-lysC) was identified and characterized. ► The expression of Ec-lysC in response to LPS, SGIV and Vibrio alginolyticus were different. ► The recombinant Ec-lysC had lytic activities against Gram-positive bacteria and Gram-negative bacteria. ► Ec-lysC overexpression decreased SGIV replication in grouper cells.</description><identifier>ISSN: 1050-4648</identifier><identifier>EISSN: 1095-9947</identifier><identifier>DOI: 10.1016/j.fsi.2012.03.027</identifier><identifier>PMID: 22579550</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>Adjuvants, Immunologic - pharmacology ; Amino Acid Sequence ; Animals ; Anti-Bacterial Agents - pharmacology ; Antimicrobial ; Antivirus ; Bacteria - drug effects ; Bass - genetics ; Bass - immunology ; c-type lysozyme ; Cells, Cultured ; Cloning, Molecular ; Epinephelus bruneus ; Epinephelus coioides ; Gene Expression Profiling ; Gene Expression Regulation - drug effects ; Gene Expression Regulation - immunology ; Head Kidney - immunology ; Iridovirus - immunology ; Lipopolysaccharides - pharmacology ; Marine ; Micrococcus lysodeikticus ; Molecular Sequence Data ; Muramidase - chemistry ; Muramidase - genetics ; Muramidase - metabolism ; Recombinant Proteins - isolation & purification ; Recombinant Proteins - pharmacology ; Sequence Alignment ; Singapore Grouper Iridovirus ; Staphylococcus aureus ; Streptococcus iniae ; Vibrio alginolyticus ; Vibrio alginolyticus - immunology</subject><ispartof>Fish & shellfish immunology, 2012-08, Vol.33 (2), p.186-196</ispartof><rights>2012 Elsevier Ltd</rights><rights>Copyright © 2012 Elsevier Ltd. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c452t-10604ae947bf19489cac8ec2da3b445ea4e86f4bdd75d2a46879ad30e6e884a23</citedby><cites>FETCH-LOGICAL-c452t-10604ae947bf19489cac8ec2da3b445ea4e86f4bdd75d2a46879ad30e6e884a23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.fsi.2012.03.027$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22579550$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wei, Shina</creatorcontrib><creatorcontrib>Huang, Youhua</creatorcontrib><creatorcontrib>Cai, Jia</creatorcontrib><creatorcontrib>Huang, Xiaohong</creatorcontrib><creatorcontrib>Fu, Jing</creatorcontrib><creatorcontrib>Qin, Qiwei</creatorcontrib><title>Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides</title><title>Fish & shellfish immunology</title><addtitle>Fish Shellfish Immunol</addtitle><description>Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and “GSTDYGIFQINS” motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.
► c-type lysozyme gene from Epinephelus coioides (Ec-lysC) was identified and characterized. ► The expression of Ec-lysC in response to LPS, SGIV and Vibrio alginolyticus were different. ► The recombinant Ec-lysC had lytic activities against Gram-positive bacteria and Gram-negative bacteria. ► Ec-lysC overexpression decreased SGIV replication in grouper cells.</description><subject>Adjuvants, Immunologic - pharmacology</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Anti-Bacterial Agents - pharmacology</subject><subject>Antimicrobial</subject><subject>Antivirus</subject><subject>Bacteria - drug effects</subject><subject>Bass - genetics</subject><subject>Bass - immunology</subject><subject>c-type lysozyme</subject><subject>Cells, Cultured</subject><subject>Cloning, Molecular</subject><subject>Epinephelus bruneus</subject><subject>Epinephelus coioides</subject><subject>Gene Expression Profiling</subject><subject>Gene Expression Regulation - drug effects</subject><subject>Gene Expression Regulation - immunology</subject><subject>Head Kidney - immunology</subject><subject>Iridovirus - immunology</subject><subject>Lipopolysaccharides - pharmacology</subject><subject>Marine</subject><subject>Micrococcus lysodeikticus</subject><subject>Molecular Sequence Data</subject><subject>Muramidase - chemistry</subject><subject>Muramidase - genetics</subject><subject>Muramidase - metabolism</subject><subject>Recombinant Proteins - isolation & purification</subject><subject>Recombinant Proteins - pharmacology</subject><subject>Sequence Alignment</subject><subject>Singapore Grouper Iridovirus</subject><subject>Staphylococcus aureus</subject><subject>Streptococcus iniae</subject><subject>Vibrio alginolyticus</subject><subject>Vibrio alginolyticus - immunology</subject><issn>1050-4648</issn><issn>1095-9947</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkU1P3DAQhi3UCijtD-il8rEHEsaO7STiVCH6IYF6gbPltSeLV1k72AnS8uvxaoFj1dOMNM-80sxDyFcGNQOmLjb1kH3NgfEamhp4e0ROGfSy6nvRftj3EiqhRHdCPuW8AQDVKDgmJ5zLtpcSTkm4jSPaZTSJ2jEGH9bUBEftg0nGzpj8s5l9DDQO1FbzbkI67nJ83m2RrjEg9WWUTFhjlac4z-joOsVlwnROrycfcHrAccnURh-9w_yZfBzMmPHLaz0j9z-v765-Vzd_f_25-nFTWSH5XDFQIAyWK1YD60XXW2M7tNyZZiWERCOwU4NYOddKx41QXdsb1wAq7DpheHNGvh9ypxQfF8yz3vpscRxNwLhkzYALpVTHm_9BG96Xx8mCsgNqU8w54aCn5Lcm7Qqk90b0Rhcjem9EQ6OLkbLz7TV-WW3RvW-8KSjA5QHA8o8nj0ln6zFYdD6hnbWL_h_xL02UnXY</recordid><startdate>20120801</startdate><enddate>20120801</enddate><creator>Wei, Shina</creator><creator>Huang, Youhua</creator><creator>Cai, Jia</creator><creator>Huang, Xiaohong</creator><creator>Fu, Jing</creator><creator>Qin, Qiwei</creator><general>Elsevier Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7T5</scope><scope>F1W</scope><scope>H94</scope><scope>H95</scope><scope>L.G</scope></search><sort><creationdate>20120801</creationdate><title>Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides</title><author>Wei, Shina ; Huang, Youhua ; Cai, Jia ; Huang, Xiaohong ; Fu, Jing ; Qin, Qiwei</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c452t-10604ae947bf19489cac8ec2da3b445ea4e86f4bdd75d2a46879ad30e6e884a23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Adjuvants, Immunologic - pharmacology</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Anti-Bacterial Agents - pharmacology</topic><topic>Antimicrobial</topic><topic>Antivirus</topic><topic>Bacteria - drug effects</topic><topic>Bass - genetics</topic><topic>Bass - immunology</topic><topic>c-type lysozyme</topic><topic>Cells, Cultured</topic><topic>Cloning, Molecular</topic><topic>Epinephelus bruneus</topic><topic>Epinephelus coioides</topic><topic>Gene Expression Profiling</topic><topic>Gene Expression Regulation - drug effects</topic><topic>Gene Expression Regulation - immunology</topic><topic>Head Kidney - immunology</topic><topic>Iridovirus - immunology</topic><topic>Lipopolysaccharides - pharmacology</topic><topic>Marine</topic><topic>Micrococcus lysodeikticus</topic><topic>Molecular Sequence Data</topic><topic>Muramidase - chemistry</topic><topic>Muramidase - genetics</topic><topic>Muramidase - metabolism</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Recombinant Proteins - pharmacology</topic><topic>Sequence Alignment</topic><topic>Singapore Grouper Iridovirus</topic><topic>Staphylococcus aureus</topic><topic>Streptococcus iniae</topic><topic>Vibrio alginolyticus</topic><topic>Vibrio alginolyticus - immunology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wei, Shina</creatorcontrib><creatorcontrib>Huang, Youhua</creatorcontrib><creatorcontrib>Cai, Jia</creatorcontrib><creatorcontrib>Huang, Xiaohong</creatorcontrib><creatorcontrib>Fu, Jing</creatorcontrib><creatorcontrib>Qin, Qiwei</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Immunology Abstracts</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><jtitle>Fish & shellfish immunology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wei, Shina</au><au>Huang, Youhua</au><au>Cai, Jia</au><au>Huang, Xiaohong</au><au>Fu, Jing</au><au>Qin, Qiwei</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides</atitle><jtitle>Fish & shellfish immunology</jtitle><addtitle>Fish Shellfish Immunol</addtitle><date>2012-08-01</date><risdate>2012</risdate><volume>33</volume><issue>2</issue><spage>186</spage><epage>196</epage><pages>186-196</pages><issn>1050-4648</issn><eissn>1095-9947</eissn><abstract>Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and “GSTDYGIFQINS” motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.
► c-type lysozyme gene from Epinephelus coioides (Ec-lysC) was identified and characterized. ► The expression of Ec-lysC in response to LPS, SGIV and Vibrio alginolyticus were different. ► The recombinant Ec-lysC had lytic activities against Gram-positive bacteria and Gram-negative bacteria. ► Ec-lysC overexpression decreased SGIV replication in grouper cells.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>22579550</pmid><doi>10.1016/j.fsi.2012.03.027</doi><tpages>11</tpages></addata></record> |
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| subjects | Adjuvants, Immunologic - pharmacology Amino Acid Sequence Animals Anti-Bacterial Agents - pharmacology Antimicrobial Antivirus Bacteria - drug effects Bass - genetics Bass - immunology c-type lysozyme Cells, Cultured Cloning, Molecular Epinephelus bruneus Epinephelus coioides Gene Expression Profiling Gene Expression Regulation - drug effects Gene Expression Regulation - immunology Head Kidney - immunology Iridovirus - immunology Lipopolysaccharides - pharmacology Marine Micrococcus lysodeikticus Molecular Sequence Data Muramidase - chemistry Muramidase - genetics Muramidase - metabolism Recombinant Proteins - isolation & purification Recombinant Proteins - pharmacology Sequence Alignment Singapore Grouper Iridovirus Staphylococcus aureus Streptococcus iniae Vibrio alginolyticus Vibrio alginolyticus - immunology |
| title | Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides |
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