Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Feiyan Wang; Amin Zhang; Xuelian Fan; Qianqian Feng; Zhizhi Zhang; Dandan Liu; Shijie Su; Zhaofeng Hou; Jinjun Xu; Xilong Kang; Zhiming Pan; Hunjie Hu; Jianping Tao

doi:10.1016/j.vetpar.2023.110060

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Vet Parasitol. 2023 Dec:324:110060. doi: 10.1016/j.vetpar.2023.110060. Epub 2023 Oct 26.

Authors

Feiyan Wang¹, Amin Zhang¹, Xuelian Fan¹, Qianqian Feng¹, Zhizhi Zhang¹, Dandan Liu¹, Shijie Su¹, Zhaofeng Hou¹, Jinjun Xu¹, Xilong Kang², Zhiming Pan³, Hunjie Hu⁴, Jianping Tao⁵

Affiliations

¹ College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China.
² Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China.
³ Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China; Suqian University, Suqian 223800, China.
⁴ School of Ecology and Environmental Sciences, Yunnan University, Kunming 650091, China.
⁵ College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China. Electronic address: jptao@yzu.edu.cn.

PMID: 37931477
DOI: 10.1016/j.vetpar.2023.110060

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 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG_-CAP was about 32 kDa 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 200 µg 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.

Keywords: Cellular invasion; Eimeria necatrix; Immunoprotection; Prokaryotic expression; SAG protein.

MeSH terms

Animals
Chickens / parasitology
Coccidiosis* / prevention & control
Coccidiosis* / veterinary
Eimeria* / physiology
Poultry Diseases* / parasitology
Recombinant Proteins / genetics
Sporozoites
Vaccines, Synthetic

Substances

Recombinant Proteins
Vaccines, Synthetic