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 (Maca ca radiata) ZP3, excluding the N-terminus signal sequence and the C-te rminus transmembrane-like domain, was amplified by PCR from a full-len gth cDNA clone. The amplified Bam HI and Sacl restricted fragment was cloned in frame downstream of the T5 promoter under lac operator centr al for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was ex pressed 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 ban d of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 wa s observed at 0.5 mM IPTG. Antisera generated in monkeys against synth etic peptides from the N-(23-45 aa residues) and C-(300-322 and 324-34 7 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequ ence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in S G13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toroid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate gener ated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum r eacted with porcine ZP3 beta and bonnet r-ZP3 but failed to react with porcine ZP3 alpha in a Western blot. Moreover, antisera when tested b y indirect immunofluorescence on bonnet monkey ovarian sections, showe d 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 immun ization in nonhuman primates. (C) 1997 Wiley-Liss, Inc.