Screening and Identification of Nanobodies Against β-Conglycinin
10.13865/j.cnki.cjbmb.2025.03.1462
- VernacularTitle:β-伴大豆球蛋白纳米抗体的筛选及鉴定
- Author:
Jia-Shu CHANG
1
;
Hua-Bo SUN
1
;
Yu-Ting WANG
1
;
Xiao-Hui WANG
1
;
Bo YANG
1
;
Hong-Rui LIU
1
;
Yue-Xin LI
1
;
Yuan-Zhao SUN
1
;
Shao-Peng GU
1
;
Jin-Xin HE
1
Author Information
1. 山西农业大学动物医学学院预防兽医系检疫检验实验室,山西 太谷 030801
- Publication Type:Journal Article
- Keywords:
soybean allergens;
β-conglycinin;
phage display library;
nanobody
- From:
Chinese Journal of Biochemistry and Molecular Biology
2025;41(5):764-770
- CountryChina
- Language:Chinese
-
Abstract:
Soy is a vital source of plant carbohydrates.However,it poses significant allergenic risks,particularly to young children and animals.Among the various proteins in soy,β-conglycinin,which con-stitutes approximately 30％of total soy carbohydrates,is a primary allergen.Undigested β-conglycinin can lead to intestinal damage by inhibiting cell growth,disrupting the cytoskeleton,and inducing apopto-sis.It can also enter the lymphatic and circulatory systems,triggering allergic reactions.Conventional ELISA methods for detecting β-conglycinin rely on polyclonal or monoclonal antibodies,which are limited by their large molecular weight,difficulty in accessing the protein core,and sensitivity to acidic and bas-ic conditions.To address these limitations,this study aimed to develop nanobodies(Nbs)against β-con-glycinin.Nbs,derived from the variable regions of heavy-chain antibodies found in camelids,have a mo-lecular weight approximately one-tenth that of conventional antibodies.They offer advantages such as small size,stable structure,high specificity,and strong affinity.A female alpacas was immunized five times using β-conglycinin,which showed a heavy chain antibody potency of 1∶16 000 by ELISA.Pe-ripheral blood lymphocytes were subsequently isolated and total RNA was extracted.The variable region of the heavy-chain antibody was amplified via PCR,and recombinant plasmids were constructed and transformed into the E.coli competency strain ER2738.The resulting library contained about 3.5×108 CFU/mL,which increased to 1.15×1012 PFU/mL after phage rescue,with a 100％Nbs gene insertion rate,indicating high diversity.Its Nbs phage output was significantly enriched by four rounds of solid-phase elution with an enrichment rate of 155.9.Four rounds of solid-phase panning yielded 35 positive clones,all of which shared the same amino acid sequence upon sequencing.The selected Nb was ex-pressed in a prokaryotic system,and its binding ability to β-conglycinin was confirmed using Western blotting and ELISA.The results demonstrated excellent specificity and affinity.This research lays the groundwork for developing a rapid and efficient detection method for β-conglycinin using Nbs,potentially enhancing food safety and allergen management.
