Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix
IntroductionApicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH_00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2).MethodsThe cDNAs...
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Frontiers Media S.A.
2023-12-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1305727/full |
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| author | Weimin Cai Weimin Cai Weimin Cai Qianqian Feng Qianqian Feng Qianqian Feng Liyue Wang Liyue Wang Liyue Wang Shijie Su Shijie Su Shijie Su Zhaofeng Hou Zhaofeng Hou Zhaofeng Hou Dandan Liu Dandan Liu Dandan Liu Xilong Kang Jinjun Xu Jinjun Xu Jinjun Xu Zhiming Pan Zhiming Pan Jianping Tao Jianping Tao Jianping Tao |
| author_facet | Weimin Cai Weimin Cai Weimin Cai Qianqian Feng Qianqian Feng Qianqian Feng Liyue Wang Liyue Wang Liyue Wang Shijie Su Shijie Su Shijie Su Zhaofeng Hou Zhaofeng Hou Zhaofeng Hou Dandan Liu Dandan Liu Dandan Liu Xilong Kang Jinjun Xu Jinjun Xu Jinjun Xu Zhiming Pan Zhiming Pan Jianping Tao Jianping Tao Jianping Tao |
| author_sort | Weimin Cai |
| collection | DOAJ |
| description | IntroductionApicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH_00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2).MethodsThe cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in Escherichia coli BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in E. necatrix, respectively. Finally, the recombinant pEGFP-C1-ΔNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein.ResultsThe EnApiAP2 gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an α-helix and three antiparallel β-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6×His tag mAb and the serum of convalescent chickens after infection with E. necatrix, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of EnApiAP2 was significantly higher in SZ than in MZ-2, third generation merozoites (MZ-3) and gametocytes (P<0.01). EnApiAP2 protein was localized in the nuclei of SZ, MZ-2 and MZ-3 of E. necatrix. The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the ΔNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein.DiscussionEnApiAP2 protein encoded by ENH_00027130 sequence was localized in the nucleus of E. necatrix parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study. |
| first_indexed | 2024-03-09T02:56:47Z |
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| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-03-09T02:56:47Z |
| publishDate | 2023-12-01 |
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| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-29055e0d01d14d449054ed5e965de5bc2023-12-05T04:21:44ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882023-12-011310.3389/fcimb.2023.13057271305727Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrixWeimin Cai0Weimin Cai1Weimin Cai2Qianqian Feng3Qianqian Feng4Qianqian Feng5Liyue Wang6Liyue Wang7Liyue Wang8Shijie Su9Shijie Su10Shijie Su11Zhaofeng Hou12Zhaofeng Hou13Zhaofeng Hou14Dandan Liu15Dandan Liu16Dandan Liu17Xilong Kang18Jinjun Xu19Jinjun Xu20Jinjun Xu21Zhiming Pan22Zhiming Pan23Jianping Tao24Jianping Tao25Jianping Tao26College of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaPrincipal's Office, Suqian University, Suqian, ChinaCollege of Veterinary Medicine, Yangzhou University, Yangzhou, ChinaJiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, ChinaJiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, ChinaIntroductionApicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH_00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2).MethodsThe cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in Escherichia coli BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in E. necatrix, respectively. Finally, the recombinant pEGFP-C1-ΔNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein.ResultsThe EnApiAP2 gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an α-helix and three antiparallel β-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6×His tag mAb and the serum of convalescent chickens after infection with E. necatrix, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of EnApiAP2 was significantly higher in SZ than in MZ-2, third generation merozoites (MZ-3) and gametocytes (P<0.01). EnApiAP2 protein was localized in the nuclei of SZ, MZ-2 and MZ-3 of E. necatrix. The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the ΔNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein.DiscussionEnApiAP2 protein encoded by ENH_00027130 sequence was localized in the nucleus of E. necatrix parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study.https://www.frontiersin.org/articles/10.3389/fcimb.2023.1305727/fullEimeriaEnApiAP2localizationNLSmAb |
| spellingShingle | Weimin Cai Weimin Cai Weimin Cai Qianqian Feng Qianqian Feng Qianqian Feng Liyue Wang Liyue Wang Liyue Wang Shijie Su Shijie Su Shijie Su Zhaofeng Hou Zhaofeng Hou Zhaofeng Hou Dandan Liu Dandan Liu Dandan Liu Xilong Kang Jinjun Xu Jinjun Xu Jinjun Xu Zhiming Pan Zhiming Pan Jianping Tao Jianping Tao Jianping Tao Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix Frontiers in Cellular and Infection Microbiology Eimeria EnApiAP2 localization NLS mAb |
| title | Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix |
| title_full | Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix |
| title_fullStr | Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix |
| title_full_unstemmed | Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix |
| title_short | Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix |
| title_sort | localization in vivo and in vitro confirms enapiap2 protein encoded by enh 00027130 as a nuclear protein in eimeria necatrix |
| topic | Eimeria EnApiAP2 localization NLS mAb |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2023.1305727/full |
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