Date of Graduation
12-2025
Document Type
Thesis
Degree Name
Master of Science in Poultry Science (MS)
Degree Level
Graduate
Department
Poultry Science
Advisor/Mentor
Sun, Xiaolun
Committee Member
Shi, Ainong
Second Committee Member
Kwon, Young Min
Keywords
B-cell epitopes; Immunoinformatic; Multiepitope fusion antigen (MEFA); Necrotic enteritis; vaccine
Abstract
Necrotic enteritis (NE), primarily induced by the Clostridium perfringens (C. perfringens), causes huge with few effective interventions. C. perfringens sporulation protein vaccine protected birds against clinical NE. In this study, we aimed to develop multiepitope fusion antigen (MEFA) vaccines carrying the representative epitopes from the sporulation vaccines. C. perfringens sporulation vaccine proteins were analyzed using proteomics. Immunogenic B-cell epitopes of the proteins were identified using immunoinformatic tools. After ELISA assay screening of two 10-mer epitopes, the selected epitopes were substituted into backbone protein of C-terminal C. perfringens enterotoxin (C-CPE) to form CPE-MEFA vaccines. After analysis of the CPE-MEFA vaccine characteristics such as epitope probability, stability, aliphatic index, hydropathicity, half-life and 3D structure, the CPE-MEFA vaccine were synthesized, subcloned, expressed, and immunoassay-tested. As a result, among 33 proteins identified in proteomics assay, 19 proteins were analyzed to identify two 10-mer B cell epitopes per protein using ElliPro and IEDB BepiPred. Subsequently, 20-mer peptides synthesized from two 10-mer epitopes were evaluated for immunogenicity using ELISA. The resultant 15 pair 10-mer epitopes were substituted into backbone C-CPE to form 36 CPE-MEFA vaccines. After evaluated for protein characteristics such as epitope probability and 3D structure, 12 CPE-MEFA vaccines were selected. Subsequent molecular dynamic simulation with GROMACS, 6 CPE-MEFA vaccines were chosen to synthesize in plasmid pET52b. After subcloning into plasmid pET28a, CPE-MEFA vaccines were expressed in BL21 cells. CPE-MEFA proteins were around 30 kda using Coomassie blue staining. Consistently, his-tag antibody Western Blot detected CPE-MEFA proteins of the same size compared to proteins from BL21 cells carrying pET28a without CPE-MEFA genes. Importantly, clear CPE-MEFA proteins at 30 kda were detected using sera from chicken immunized with C. perfringens sporulation protein vaccines. Together, our results show that CPE-MEFA vaccines have been constructed, expressed, and evaluated. The immunogenic Western Blot results suggest that the CPE-MEFA vaccines have the potential to prevent chicken NE.
Citation
Shrestha, J. (2025). Developing Multiepitope Fusion Antigen Vaccines of Clostridium perfringens Against Chicken Necrotic Enteritis. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/5979
