Preparation and identification human anti-Siglec-9 Fab fragment antibody
10.3969/j.issn.1000-484X.2018.06.015
- VernacularTitle:人源化抗Siglec-9抗体Fab片段的制备及鉴定
- Author:
Sa-Sa CHU
1
;
Na YOU
;
Xin ZHANG
;
Zhi-Guo YANG
;
Jin ZHU
;
Mao-Rong WANG
Author Information
1. 南京中医药大学附属八一医院
- Keywords:
Siglec-9;
Fab fragment;
Human anti-Siglec-9 antibody;
THP-1 cell;
Inflammatory cytokines
- From:
Chinese Journal of Immunology
2018;34(6):877-881
- CountryChina
- Language:Chinese
-
Abstract:
Objective:The prokaryotic expression vector of human anti-Siglec-9 Fab fragment antibody was constructed and purified,while was identified. Methods:The variable and conserved regions of heavy chain and light chain were obtained by polymerase chain reaction respectively(PCR),which was combined by overlap extension PCR and was digested with restriction enzyme,and then it was transformed into Escherichia coli BL21 and was purified by His-trap Lambda Fab column and AKTA system. SDS-PAGE,ELISA and Western blot were used for the identification of human anti-Siglec-9 Fab fragment antibody. The effect of human anti-Siglec-9 Fab fragment antibody on regulating the mRNA expression of TNF-α,IL-1,IL-6,IL-8 was detected by real-time PCR. Results:Successfully obtained the chains of heavy and light, while constructed an activation human anti-Siglec-9 Fab fragment antibody which could specifically bind to Siglec-9 protein. The human anti-Siglec-9 Fab fragment antibody could specifically bind to Siglec-9 was confirmed by SDS-PAGE,ELISA and Western blot. The human anti-Siglec-9 Fab fragment antibody inhibited the mRNA expression of TNF-α,IL-1, IL-6,IL-8. Conclusion:Successful prokaryotic expression,purification,character analysis,and suppressed the mRNA expression of in-flammatory cytokines with the human anti-Siglec-9 Fab fragment antibody and lay the biology foundation for the further study.
