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Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection
Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, litt...
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| Published in: | Veterinary parasitology 2023-12, Vol.324, p.110060-110060, Article 110060 |
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| creator | Wang, Feiyan Zhang, Amin Fan, Xuelian Feng, Qianqian Zhang, Zhizhi Liu, Dandan Su, Shijie Hou, Zhaofeng Xu, Jinjun Kang, Xilong Pan, Zhiming Hu, Hunjie Tao, Jianping |
| description | Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813bp, coding 270 amino acids with a predicated molecular weight of 28.86kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P |
| doi_str_mv | 10.1016/j.vetpar.2023.110060 |
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•A novel E. necatrix sag gene was cloned, encoding a protein with a CAP domain.•EnSAG is anchored to the membrane of parasites via a GPI anchor•EnSAG might participate in sporozoite invasion into MDBK cells.•EnSAG could be used as a candidate antigen to develop a recombinant coccidiosis vaccine.</description><identifier>ISSN: 0304-4017</identifier><identifier>EISSN: 1873-2550</identifier><identifier>DOI: 10.1016/j.vetpar.2023.110060</identifier><language>eng</language><publisher>Elsevier B.V</publisher><subject>antigens ; Bacillus cereus ; blood serum ; Cellular invasion ; chickens ; coccidiosis ; domain ; Eimeria necatrix ; enzymatic reactions ; fluorescent antibody technique ; genes ; Immunoprotection ; membrane proteins ; merozoites ; molecular weight ; monoclonal antibodies ; pathogens ; phospholipase C ; polyclonal antibodies ; poultry industry ; Prokaryotic expression ; quantitative polymerase chain reaction ; recombinant proteins ; SAG protein ; sporozoites ; vaccines ; veterinary parasitology ; Western blotting</subject><ispartof>Veterinary parasitology, 2023-12, Vol.324, p.110060-110060, Article 110060</ispartof><rights>2023</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c372t-1b661d3342f01b08bbe438043ff4a44fb526fbdd3a3be20b8c92a35c9c4553233</citedby><cites>FETCH-LOGICAL-c372t-1b661d3342f01b08bbe438043ff4a44fb526fbdd3a3be20b8c92a35c9c4553233</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids></links><search><creatorcontrib>Wang, Feiyan</creatorcontrib><creatorcontrib>Zhang, Amin</creatorcontrib><creatorcontrib>Fan, Xuelian</creatorcontrib><creatorcontrib>Feng, Qianqian</creatorcontrib><creatorcontrib>Zhang, Zhizhi</creatorcontrib><creatorcontrib>Liu, Dandan</creatorcontrib><creatorcontrib>Su, Shijie</creatorcontrib><creatorcontrib>Hou, Zhaofeng</creatorcontrib><creatorcontrib>Xu, Jinjun</creatorcontrib><creatorcontrib>Kang, Xilong</creatorcontrib><creatorcontrib>Pan, Zhiming</creatorcontrib><creatorcontrib>Hu, Hunjie</creatorcontrib><creatorcontrib>Tao, Jianping</creatorcontrib><title>Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection</title><title>Veterinary parasitology</title><description>Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813bp, coding 270 amino acids with a predicated molecular weight of 28.86kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P <0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.
[Display omitted]
•A novel E. necatrix sag gene was cloned, encoding a protein with a CAP domain.•EnSAG is anchored to the membrane of parasites via a GPI anchor•EnSAG might participate in sporozoite invasion into MDBK cells.•EnSAG could be used as a candidate antigen to develop a recombinant coccidiosis vaccine.</description><subject>antigens</subject><subject>Bacillus cereus</subject><subject>blood serum</subject><subject>Cellular invasion</subject><subject>chickens</subject><subject>coccidiosis</subject><subject>domain</subject><subject>Eimeria necatrix</subject><subject>enzymatic reactions</subject><subject>fluorescent antibody technique</subject><subject>genes</subject><subject>Immunoprotection</subject><subject>membrane proteins</subject><subject>merozoites</subject><subject>molecular weight</subject><subject>monoclonal antibodies</subject><subject>pathogens</subject><subject>phospholipase C</subject><subject>polyclonal antibodies</subject><subject>poultry industry</subject><subject>Prokaryotic expression</subject><subject>quantitative polymerase chain reaction</subject><subject>recombinant proteins</subject><subject>SAG protein</subject><subject>sporozoites</subject><subject>vaccines</subject><subject>veterinary parasitology</subject><subject>Western blotting</subject><issn>0304-4017</issn><issn>1873-2550</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><recordid>eNqFkU1LAzEQhoMoWD_-gYccvWydfO1uL0Ip9QMEBfUcstkJpnQ3NdlW_ffGrmc9DbzzzjvMPIRcMJgyYOXVarrDYWPilAMXU8YASjggE1ZXouBKwSGZgABZSGDVMTlJaQUAEspqQt6Xn5uIKfnQ0-Cooc_zW7qJYUDf0w8_vGVpMX-ibehMVlwMHV36DqM3tEdrhug_qelb6odEY1gjzS7f78w-cd_oum0f9pF2yOIZOXJmnfD8t56S15vly-KueHi8vV_MHworKj4UrClL1gohuQPWQN00KEUNUjgnjZSuUbx0TdsKIxrk0NR2xo1QdmalUoILcUoux9y8-n2LadCdTxbXa9Nj2CYtmJJMVTMG_1p5XZczUalKZqscrTaGlCI6vYm-M_FLM9A_MPRKjzD0Dww9wshj1-MY5ot3HqNO1mNvsfUxv0W3wf8d8A1kZpR2</recordid><startdate>20231201</startdate><enddate>20231201</enddate><creator>Wang, Feiyan</creator><creator>Zhang, Amin</creator><creator>Fan, Xuelian</creator><creator>Feng, Qianqian</creator><creator>Zhang, Zhizhi</creator><creator>Liu, Dandan</creator><creator>Su, Shijie</creator><creator>Hou, Zhaofeng</creator><creator>Xu, Jinjun</creator><creator>Kang, Xilong</creator><creator>Pan, Zhiming</creator><creator>Hu, Hunjie</creator><creator>Tao, Jianping</creator><general>Elsevier B.V</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7S9</scope><scope>L.6</scope></search><sort><creationdate>20231201</creationdate><title>Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection</title><author>Wang, Feiyan ; Zhang, Amin ; Fan, Xuelian ; Feng, Qianqian ; Zhang, Zhizhi ; Liu, Dandan ; Su, Shijie ; Hou, Zhaofeng ; Xu, Jinjun ; Kang, Xilong ; Pan, Zhiming ; Hu, Hunjie ; Tao, Jianping</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c372t-1b661d3342f01b08bbe438043ff4a44fb526fbdd3a3be20b8c92a35c9c4553233</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>antigens</topic><topic>Bacillus cereus</topic><topic>blood serum</topic><topic>Cellular invasion</topic><topic>chickens</topic><topic>coccidiosis</topic><topic>domain</topic><topic>Eimeria necatrix</topic><topic>enzymatic reactions</topic><topic>fluorescent antibody technique</topic><topic>genes</topic><topic>Immunoprotection</topic><topic>membrane proteins</topic><topic>merozoites</topic><topic>molecular weight</topic><topic>monoclonal antibodies</topic><topic>pathogens</topic><topic>phospholipase C</topic><topic>polyclonal antibodies</topic><topic>poultry industry</topic><topic>Prokaryotic expression</topic><topic>quantitative polymerase chain reaction</topic><topic>recombinant proteins</topic><topic>SAG protein</topic><topic>sporozoites</topic><topic>vaccines</topic><topic>veterinary parasitology</topic><topic>Western blotting</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wang, Feiyan</creatorcontrib><creatorcontrib>Zhang, Amin</creatorcontrib><creatorcontrib>Fan, Xuelian</creatorcontrib><creatorcontrib>Feng, Qianqian</creatorcontrib><creatorcontrib>Zhang, Zhizhi</creatorcontrib><creatorcontrib>Liu, Dandan</creatorcontrib><creatorcontrib>Su, Shijie</creatorcontrib><creatorcontrib>Hou, Zhaofeng</creatorcontrib><creatorcontrib>Xu, Jinjun</creatorcontrib><creatorcontrib>Kang, Xilong</creatorcontrib><creatorcontrib>Pan, Zhiming</creatorcontrib><creatorcontrib>Hu, Hunjie</creatorcontrib><creatorcontrib>Tao, Jianping</creatorcontrib><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><jtitle>Veterinary parasitology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wang, Feiyan</au><au>Zhang, Amin</au><au>Fan, Xuelian</au><au>Feng, Qianqian</au><au>Zhang, Zhizhi</au><au>Liu, Dandan</au><au>Su, Shijie</au><au>Hou, Zhaofeng</au><au>Xu, Jinjun</au><au>Kang, Xilong</au><au>Pan, Zhiming</au><au>Hu, Hunjie</au><au>Tao, Jianping</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection</atitle><jtitle>Veterinary parasitology</jtitle><date>2023-12-01</date><risdate>2023</risdate><volume>324</volume><spage>110060</spage><epage>110060</epage><pages>110060-110060</pages><artnum>110060</artnum><issn>0304-4017</issn><eissn>1873-2550</eissn><abstract>Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813bp, coding 270 amino acids with a predicated molecular weight of 28.86kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P <0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.
[Display omitted]
•A novel E. necatrix sag gene was cloned, encoding a protein with a CAP domain.•EnSAG is anchored to the membrane of parasites via a GPI anchor•EnSAG might participate in sporozoite invasion into MDBK cells.•EnSAG could be used as a candidate antigen to develop a recombinant coccidiosis vaccine.</abstract><pub>Elsevier B.V</pub><doi>10.1016/j.vetpar.2023.110060</doi><tpages>1</tpages></addata></record> |
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| source | ScienceDirect Freedom Collection |
| subjects | antigens Bacillus cereus blood serum Cellular invasion chickens coccidiosis domain Eimeria necatrix enzymatic reactions fluorescent antibody technique genes Immunoprotection membrane proteins merozoites molecular weight monoclonal antibodies pathogens phospholipase C polyclonal antibodies poultry industry Prokaryotic expression quantitative polymerase chain reaction recombinant proteins SAG protein sporozoites vaccines veterinary parasitology Western blotting |
| title | Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection |
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