Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding.
To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.
Sivapurapu
Neela
N
Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.
Upadhyay
Abhishek
A
Hasegawa
Akiko
A
Koyama
Koji
K
Gupta
Satish K
SK
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001483:Base Sequence; D004527:Egg Proteins / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D005260:Female; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D013084:Sperm-Ovum Interactions / Q000187:drug effects / Q000502:physiology*; D000072216:Zona Pellucida Glycoproteins
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004527:Egg Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D000072216:Zona Pellucida Glycoproteins