[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].
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.
Song
Li-Wen
LW
Shanghai Medical College Fudan University, Shanghai 200032, China.
Wang
Yu-Bao
YB
Ni
Ya
Y
He
Ya-Ping
YP
Hong
Ai-Zhen
AZ
Hinsch
Elvira
E
Hinsch
Klaus-Dieter
KD
Chow
Si-Chang
SC
Yuan
Yu-Ying
YY
Shi
Qi-Xian
QX
Xu
Wan-Xiang
WX
D000818:Animals; D004527:Egg Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology*; D007114:Immunization; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D013084:Sperm-Ovum Interactions / Q000276:immunology*; D000072216:Zona Pellucida Glycoproteins
D004527:Egg Proteins; D007106:Immune Sera; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606517:ZP3 protein, human; C082833:ZP4 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse