Prokaryotic expression of mouse LRP16, preparation and identification of rabbit anti-mouse LRP16 polyclonal antibody.
- Author:
Feifei ZHANG
1
;
Jian LI
1
;
Xiangying XU
1
;
Meiling HAN
1
;
Zhe ZHANG
2
Author Information
1. College of Anesthesiology, Xuzhou Medical University, Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou 221004, China.
2. College of Anesthesiology, Xuzhou Medical University, Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou 221004, China. *Corresponding author, E-mail: zhangzhe70@xzhmu.edu.cn.
- Publication Type:Journal Article
- MeSH:
Animals;
Rabbits;
Mice;
Antibodies/immunology*;
Escherichia coli/metabolism*;
Enzyme-Linked Immunosorbent Assay;
Blotting, Western;
Antibody Specificity
- From:
Chinese Journal of Cellular and Molecular Immunology
2025;41(6):544-551
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate prokaryotic expression of the antigen sequence (amino acids 59-145) of mouse leukemia-related protein 16 (LRP16) protein and preparation of rabbit anti-mouse LRP16 polyclonal antibody. Methods The prokaryotic expression plasmid pLS962-LRP16 was constructed by the molecular cloning method and transferred into E.coli Rosetta to express LRP16 protein induced by IPTG. The recombinant protein was purified using Ni-NTA affinity columns followed by gel filtration chromatography. New Zealand white rabbits were immunized with the purified antigen to generate polyclonal antiserum, with antibody titer quantified by ELISA. Antigen-specific IgG was affinity-purified using Sepharose-coupled LRP16 and validated through Western blot and immunofluorescence assays. Results SDS-PAGE analysis confirmed insoluble expression of the LRP16 fusion protein as inclusion bodies. ELISA demonstrated exceptional antiserum titer (1:256 000). Western blot and immunofluorescence verified that the polyclonal antibody could specifically recognize endogenous LRP16 in murine tissues. Conclusion The prokaryotic expression of the LRP16 gene is successfully achieved, and the rabbit anti-mouse LRP16 polyclonal antibody exhibiting high specificity is prepared. This lays the foundation for further studies on the function of the LRP16 gene.
