Bioinformatics analysis of ureaplasma urealyticum UP3-RS02445 and the preparation of monoclonal antibodies.
- Author:
Hengxin CHEN
1
;
Xiaohui JIA
1
;
Yahui LI
2
;
Yan ZHOU
1
;
Tianjun JIA
1
;
Ping LI
3
,
4
Author Information
1. Key Laboratory of Clinical Laboratory and Diagnostics, Hebei North University, Zhangjiakou 075000, China.
2. Pathogenic Biology and Immunology, Hebei North University, Zhangjiakou 075000, China.
3. Key Laboratory of Clinical Laboratory and Diagnostics, Hebei North University, Zhangjiakou 075000, China. *Corresponding author, E-mail: zjkliping@
4. com.
- Publication Type:Journal Article
- MeSH:
Antibodies, Monoclonal/immunology*;
Animals;
Mice, Inbred BALB C;
Female;
Computational Biology/methods*;
Mice;
Ureaplasma urealyticum/genetics*;
Bacterial Proteins/genetics*;
Antibody Specificity;
Enzyme-Linked Immunosorbent Assay;
Hybridomas/immunology*
- From:
Chinese Journal of Cellular and Molecular Immunology
2024;40(11):1011-1017
- CountryChina
- Language:Chinese
-
Abstract:
Objective To make the bioinformatics analysis of Ureaplasma parvum UP3-RS02445 and prepare monoclonal antibody (mAb) against UP3-RS02445. Methods The biological characteristics of UP3-RS02445 protein were predicted by bioinformatics software. The UP3-RS02445 prokaryotic expression plasmid was constructed and the corresponding protein expression was induced by isopropyl-β-D-thiogalactoside (IPTG). Thus the expressed protein was used as immunogen to immunize female BALB/c mice. Hybridoma cell technology was used to prepare the monoclonal antibody against UP3-RS02445. The specificity and titer of monoclonal antibody were detected by Western blot and ELISA respectively. The subclass of heavy chain and subtype of light chain were identified by monoclonal antibody subtype identification test strip. Results Bioinformatics analysis showed that UP3-RS02445 protein was composed of 201 amino acids, without transmembrane domain and signal peptide, and belongs to non-secretory proteins. The recombinant prokaryotic plasmid of UP3-RS02445 was successfully constructed and the recombinant protein could be induced in large amount. After cell fusion, two hybridoma cells (A1H5 and A4E2) secreting UP3-RS02445 mAb were screened by ELISA and Western blot. The results of ELISA showed that the titers of monoclonal antibodies were 1:2560. Western blot and Immunofluorescence technique both indicated that the antibodies could bind specifically to the UP3-RS02445 protein. The heavy chain and light chain of the two mAbs were IgG1 and kappa subtypes respectively. Conclusion We prepared the UP3-RS02445 monoclonal antibodies with well specificity and high titer which might lay foundations for the subsequent development of UP diagnostic reagents and the functional study of protein.
