Immunogenicity of recombinant human zona pellucida-3 peptides expressed in E. coli and efficacy of their antisera to inhibit in vitro human sperm-egg binding.
- Author:
Li-Wen SONG
1
;
Yu-Bao WANG
;
Ya NI
;
Ya-Ping HE
;
Ai-Zhen HONG
;
Elvira HINSCH
;
Klaus-Dieter HINSCH
;
Si-Chang CHOW
;
Yu-Ying YUAN
;
Qi-Xian SHI
;
Wan-Xiang XU
Author Information
1. Shanghai Medical College Fudan University, Shanghai 200032, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Egg Proteins;
biosynthesis;
genetics;
immunology;
Escherichia coli;
genetics;
metabolism;
Female;
Humans;
Immune Sera;
immunology;
Immunization;
Male;
Membrane Glycoproteins;
biosynthesis;
genetics;
immunology;
Rabbits;
Receptors, Cell Surface;
biosynthesis;
genetics;
immunology;
Recombinant Proteins;
biosynthesis;
genetics;
immunology;
Sperm-Ovum Interactions;
immunology;
Zona Pellucida Glycoproteins
- From:
Acta Physiologica Sinica
2005;57(6):682-688
- CountryChina
- Language:Chinese
-
Abstract:
The present study was aimed to analyze the immunogenicity of recombinant human zona pellucida-3 peptides (r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348)) expressed in E. coli through immunizing rabbits, and to evaluate the efficacy of their polyclonal antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding respectively. Male New Zealand rabbits were immunized using r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) as antigen respectively, which was purified through an improved method of preparative gel polyacryulamide gel electrophoresis. The antibody response level of r-huZP3a(22 approximately 176) or r-huZP3b(177 approximately 348) immunogen in rabbits was determined by ELISA using mouse ZP3-5 (amino acid sequence(137 approximately 150) being completely conserved with huZP3(138 approximately 151) sequence) and specific huZP3-14 (amino acid sequence(327 approximately 340)) synthetic peptides as coating antigens respectively. The immunoreactivity and specificity of the anti-r-huZP3a(22 approximately 176) and anti-r-huZP3b(177 approximately 348) antisera with each r-huZP3 peptides, were tested by immunoblot and immunohistochemistry (using native huZP and human ovary section) respectively. A competitive hemizona assay (HZA) was used to evaluate the efficacy of the antisera against r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) to inhibit in vitro human sperm-egg binding. Both r-huZP3 peptides were able to induce higher antibody titers in rabbits. Each antiserum could specifically recognize or bind to each target r-huZP3 peptide expressed in E. coli and native human ZP in vitro. The antisera also inhibited sperm-egg binding in the HZA. These results show that r-huZP3a(22 approximately 176) and r-huZP3b(177 approximately 348) are of strong immunogenicity. They can be used to develop a kit for detecting whether there are autoantibodies to zona pellucida in unexplained infertile women, and their antisera might be useful tools for determining minimal B-cell epitope sequences of several known huZP3 epitope peptides.
