Establishment of single-chain antibody library targeting canine NT-proCNP,and screening and immune activity detection of a selected single-chain antibody
10.16303/j.cnki.1005-4545.2025.03.19
- VernacularTitle:犬NT-proCNP单链抗体库的构建及1株单链抗体的筛选与免疫活性检测
- Author:
Shaojia JIANG
1
;
Sha NAN
1
;
Huikang WANG
1
;
Ling MAO
1
;
Ruiling YIN
1
;
Qianghui LEI
1
;
Haolong WANG
1
;
Hao LI
1
;
Jinyu XIAO
1
;
Mingxing DING
1
;
Yi DING
1
Author Information
1. 华中农业大学动物科学技术学院/动物医学院,湖北武汉 430000
- Publication Type:Journal Article
- Keywords:
NT-proCNP;
scFv;
phage display
- From:
Chinese Journal of Veterinary Science
2025;45(3):535-541
- CountryChina
- Language:Chinese
-
Abstract:
The amino-terminal pro-C-type natriuretic peptide(NT-proCNP)is a diagnostic inflam-matory marker clinically used for diagnosing bacterial infections.This study aims to establish a phage display library of single-chain variable fragment(scFv)antibodies against canine NT-proC-NP and to screen for scFvs with high binding affinity to NT-proCNP.Initially,NT-proCNP was prepared using prokaryotic expression system and was used to immunize New Zealand White rab-bits.Upon achieving the desired serum titer,total RNA was extracted from the splenocytes of rab-bits and reverse transcribed into cDNA.Using this cDNA as a template,degenerate primers were employed to amplify the genes of the rabbit antibody light chain variable region(VL)and heavy chain variable region(VH).The VL and VH regions were spliced together to form a complete scFv fragment via overlap extension PCR.The scFv was then ligated into the phagemid pComb3XSS and electroporated into competent E.coli TG1 cells to construct a rabbit-derived anti-NT-proCNP scFv immunological library.This library underwent four rounds of enrichment and screening to isolate specific single-chain antibodies.The selected antibody was subsequently ex-pressed in a soluble form within a prokaryotic system,and its immunological activity was evalua-ted.Using phage display technology,this study successfully identified a single-chain antibody scFv-1-CNP with strong antigen-binding activity and genetic sequence characteristics of scFvs,providing a research direction for further exploration of scFv applications in the detection of NT-proCNP.
