Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity

Abstract

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 Sacl restricted fragment was cloned in frama 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 ZP3β—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 ZP3β and bonnet r‐ZP3 but failed to react with porcine ZP3α 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. Mol. Reprod. Dev. 47:140–147, 1997. © 1997 Wiley‐Liss, Inc.

Evaluation of a DNA Vaccine for Immunocontraceptive Potential Against Zona Pellucida Glycoproteins in Cattle

“…These results are in accordance with the previously described research of Turner et al [2] , in which progesterone production was depleted over time in response to ZP immunization. It is therefore evident that the cows that were given DNA encoded with ZP55 and ZP75 were affected by immunization and decreased their abilities to produce CL.…”

Prospect for Immunocontraception using the NH2‐terminal Recombinant Peptide of Human Zona Pellucida Protein (hZPA)

“…We have also observed that a synthetic peptide composed of 18 amino acids of completely identical amino acid sequence produced an autoantibody reactive to zona pellucida in rabbits 37 . In agreement with our findings, other researchers reported that recombinant peptides of homologous sequence evoked antibodies reactive to self‐zona pellucida 38 , 39 . It suggested that B‐cell clones recognizing self‐zona pellucida antigens are not completely eliminated in animals.…”

“…In the past 10 years, the immunogenicities of various recombinant zona pellucida proteins were tested including rabbit ZPA, ZPB, 40–42 monkey ZPA, ZPB, ZPC, 38 , 39 ,47,48 and human ZPA, ZPC 26 , 30 . The recombinant rabbit ZPA and ZPB produced in E. coli.…”

Mapping of Minimal Motifs of B-Cell Epitopes on Human Zona Pellucida Glycoprotein-3

“…At present, we cannot explain these distinct mapping results, including the abovementioned 8mer peptide 334-341 , because both groups employed different antisera (against r-hZP3 22-460 and r-hZP3a , resp.). The antigenicity and immunogenicity of hZP3 172-190 [17], mstZP3 301-320 [18,26], and bmZP3 300-322 [27] were previously reported. These synthetic peptides elicited antibodies capable of reacting to native mstZP, bmZP, and hZP, respectively.…”