Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein

Abstract

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for an immunocontraceptive vaccine. The efficacy of such a vaccine has to be evaluated in nonhuman primates, thus necessitating the characterization of their ZP glycoproteins. A bonnet monkey (Macaca radiata) ovarian cDNA Λgt11 library was screened for ZP2 (bZP2) using full-length human ZP2 cDNA as a probe. Two identical full-length clones with an open reading frame of 2235 nt encoding a polypeptide of 745 aa residues were isolated. The deduced aa sequence of bZP2 revealed high sequence identity (94.2%) with human ZP2. The bZP2 cDNA (115–1914 nt, 1.8 kb), excluding sequences coding for N-terminal signal sequence and C-terminal transmembranelike domain, was PCR amplified and Sac1-Sal1 restricted fragment cloned in frame downstream of the T5 promoter under the lac operator control in a pQE-30 vector. Recombinant bZP2 (r-bZP2) was expressed as a polyhistidine fusion protein in Escherichia coli strain M15 [pREP4]. Immunoblot with rabbit polyclonal antibodies against bZP2 synthetic peptide (corresponding to aa residues 429-444; K₄₃₄ replaced by R and I₄₃₆ by V) revealed a major band of 68 kDa. Immunization of male rabbits with the r-bZP2 protein purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with r-bZP2 in ELISA as well as with native protein as revealed by positive fluorescence of ZP of bonnet monkey ovary. The availability of r-bZP2 and its aa sequence will help in the development and evaluation of a contraceptive vaccine based on ZP2.