Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity.
An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone. The amplified Bam HI and SacI restricted fragment was cloned in frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strains SG13009[pREP4] and BL-21(DE3). Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3 beta-a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 was observed at 0.5 mM IPTG. Antisera generated in monkeys against synthetic peptides from the N-(23-45 aa residues) and C-(300-322 and 324-347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in SG13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum reacted with porcine ZP3 beta and bonnet r-ZP3 but failed to react with porcine ZP3 alpha in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates.
Kaul
R
R
Gamete Antigen Laboratory, National Institute of Immunology, New Delhi, India.
Afzalpurkar
A
A
Gupta
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D003269:Contraception, Immunologic; D004168:Diphtheria Toxoid / Q000276:immunology; D004527:Egg Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli; D005260:Female; D005796:Genes; D006031:Glycosylation; D000882:Haplorhini / Q000235:genetics* / Q000276:immunology; D007114:Immunization; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D010053:Ovary / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011817:Rabbits / Q000276:immunology; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D013045:Species Specificity; D013552:Swine / Q000276:immunology; D014614:Vaccines, Synthetic / Q000276:immunology; D000072216:Zona Pellucida Glycoproteins
D004168:Diphtheria Toxoid; D004527:Egg Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D000072216:Zona Pellucida Glycoproteins