Development of a shark single-domain antibody targeting a unique B cell epitope in the SARS-CoV-2 spike protein
10.3969/j.issn.1002-2694.2025.00.016
- VernacularTitle:靶向SARS-CoV-2棘突蛋白中独特B细胞抗原表位的鲨鱼单域抗体的研制
- Author:
Yue WANG
1
;
Li-jun SHEN
;
Quan FANG
;
Feng ZHANG
;
Yong-neng LUO
Author Information
1. 杭州医学院 基础医学与法医学院,杭州 310013
- Publication Type:Journal Article
- Keywords:
SARS-CoV-2;
spike protein;
B cell epitope;
Chiloscy llium plagiosum;
single domain antibody
- From:
Chinese Journal of Zoonoses
2025;41(1):32-39
- CountryChina
- Language:Chinese
-
Abstract:
The purpose of this study is to develop a shark single domain antibody(SdAb)targeting a unique B cell epitope in the SARS-CoV-2 spike protein,and explore its role in the immunological detection targeting SARS-CoV-2 spike protein S.A u-nique peptide S9 was artificially synthesized based on the sequence of a unique B cell epitope of SARS-CoV-2 spike protein,then it was conjugated to the carrier protein KLH.It was used as an immunogen for subcutaneous injection into shark back and boosted according to the standard immunization protocol.Blood collected from shark tail vein and peripheral blood lymphocytes(PBL)were isolated.Total RNA was purified from PBL and transcribed to cDNA by reverse transcription.Shark vNAR frag-ments were amplified from cDNA templates and cloned into pComb3XSS vector to obtain phage library.A positive clone named T01 was obtained through screening the phage library by indirect ELISA.Then its gene was cloned into the expression vector pET-28a.The SdAb T01 was then prokaryotically expressed and purified,and its specific recognition of the SARS-CoV-2 spike protein S was indentified by Western-blot(WB),indirect ELISA and IF A.T01 binds well with peptide S9 at EC50 value of 2.050±0.064 nmol/L.The purified SdAb T01 was proven by WB to be able to selectively detect recombinant spike protein sub-unit 1(S1)of SARS-CoV-2,with no cross-reactive to recombinant spike protein subunit 1 of other six human coronavirus.It was showed by ELISA that SdAb T01 can sensitively detect the recombinant N terminal domain(NTD)of SARS-CoV-2 pro-tein.Moreover,it also specifically recognizes the spike protein of SARS-CoV-2 that was transiently expressed in transfected HEK293 cells by IFA.Therefore,a shark single domain antibody targeting a unique B cell epitope in the spike protein of SARS-CoV-2 was successfully developed,and has shown potential immunodiagnostic value by WB,ELISA and IFA.Thus,it provides an effective tool for unique antigen detection of SARS-CoV-2.
