Preparation of Pancreatic Polypeptide Nanobody and the Analysis of Binding Activity
10.13865/j.cnki.cjbmb.2024.07.1111
- VernacularTitle:胰腺多肽纳米抗体的制备及结合活性分析
- Author:
Hong-Rui REN
1
;
Qiong JIA
;
Jia-Qin WANG
;
Jing-Jing TIAN
;
Rong-Jie LI
;
Hua-Hua HAO
;
Jian-Li LI
;
Zhi-Can QU
;
Rui-Wen FAN
Author Information
1. 山西农业大学动物医学学院羊驼生物工程实验室,山西晋中 030801
- Keywords:
pancreatic polypeptide(PP);
nanobody;
prokaryotic expression;
binding activity
- From:
Chinese Journal of Biochemistry and Molecular Biology
2024;40(9):1273-1281
- CountryChina
- Language:Chinese
-
Abstract:
Pancreatic polypeptide(PP),a pancreatic hormone containing 36 amino acids,plays impor-tant roles in the diagnosis and evaluation of pancreatic function,injury and diseases.In this study,a phage nanobody library against PP was constructed to screen specific PP nanobodies,which would be used to evaluate whether they have binding activity with PP antigen.After PP antigen with high purity was prepared by prokaryotic expression system,it was used to immunize alpaca to construct the nanobody li-brary against PP with high storage capacity and high abundance,from which 8 strains of PP nanobodies were obtained by phage display.One of nanobody strain(PP-VHH)was selected to be expressed in a prokaryotic expression system,which was induced overnight by IPTG.After purification and identifica-tion,the antigen-antibody binding activity and PP level in serum were detected by indirect ELISA and Sandwich ELISA methods,respectively.The results showed that PP-VHH had binding activity with PP,which could be used to detect PP in chicken and human serum.The Sandwich ELISA methods with R2 of the fitting curve 0.9868 could be used to detect PP concentrations of 48-55 pg/mL in the serum of chick-ens,while the concentrations of PP in human serum varied significantly.In summary,PP-VHH screened from nanobody library against PP could detect PP in serum,which would supply the basis for evaluation of abnormal pancreatic function and diagnosis of relative disease.
