Improving the immunogenicity and protective efficacy of the EtMIC2 protein against Eimeria tenella infection through random mutagenesis |
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Affiliation: | 1. Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;2. National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;3. College of Animal Science and Technology, Hebei North University, Zhangjiakou 075000, China |
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Abstract: | In recent years, directed evolution has emerged as an efficient tool to develop and identify novel protein variants. Eimeria tenella microneme-2 (EtMIC2) is a promising vaccine candidate for use against E. tenella infection; however, it only yields partial protection. The present study aimed to improve the immunogenicity and protective efficacy of EtMIC2 through random mutagenesis. Mutagenesis gene libraries of EtMIC2 were generated using error-prone polymerase chain reaction (epPCR), and the corresponding variant proteins were displayed on the yeast cell surface. Variant EtMIC2 proteins with high immunogenicity were screened through fluorescence-activated cell sorting (FACS) based on the affinity between polyclonal antibodies and antigens. Seven effective variant proteins were screened out and heterogeneously expressed in Escherichia coli as subunit vaccines. The protective efficacy of the variant proteins against E. tenella infections was then evaluated in chicken. Two variant proteins (1130 and 2119) displayed higher immunogenicity and protective efficacy than the wild-type EtMIC2 protein against E. tenella infections, increasing body weight gains and significantly decreasing lesion scores and fecal oocyst shedding, and increasing sIgA antibody production and lymphocyte proliferation. These variants displayed potential for use in the development of subunit vaccines for coccidiosis in chickens. The present results also indicate that directed evolution technology is useful for improving the immunogenicity and protective efficacy of parasite antigens. |
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Keywords: | Random mutagenesis Yeast surface display Fluorescence-activated cell sorting Immunogenicity EtMIC2" },{" #name" :" keyword" ," $" :{" id" :" k0040" }," $$" :[{" #name" :" text" ," $$" :[{" #name" :" italic" ," _" :" Eimeria tenella" },{" #name" :" __text__" ," _" :" microneme-2 epPCR" },{" #name" :" keyword" ," $" :{" id" :" k0050" }," $$" :[{" #name" :" text" ," _" :" error-prone polymerase chain reaction FACS" },{" #name" :" keyword" ," $" :{" id" :" k0060" }," $$" :[{" #name" :" text" ," _" :" fluorescence activated cell sorting MFI" },{" #name" :" keyword" ," $" :{" id" :" k0070" }," $$" :[{" #name" :" text" ," _" :" mean fluorescent intensity FCM" },{" #name" :" keyword" ," $" :{" id" :" k0080" }," $$" :[{" #name" :" text" ," _" :" flow cytometry CFA" },{" #name" :" keyword" ," $" :{" id" :" k0090" }," $$" :[{" #name" :" text" ," _" :" complete Freund’s adjuvant IFA" },{" #name" :" keyword" ," $" :{" id" :" k0100" }," $$" :[{" #name" :" text" ," _" :" incomplete Freund’s adjuvant ACI" },{" #name" :" keyword" ," $" :{" id" :" k0110" }," $$" :[{" #name" :" text" ," _" :" anti-coccidial index PBLs" },{" #name" :" keyword" ," $" :{" id" :" k0120" }," $$" :[{" #name" :" text" ," _" :" peripheral blood lymphocytes OD" },{" #name" :" keyword" ," $" :{" id" :" k0130" }," $$" :[{" #name" :" text" ," _" :" optical density |
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