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Expression of bonnet monkey ( Macaca radiata ) zona pellucida‐3 (ZP3) in a prokaryotic system and its immunogenicity
Author(s) -
Kaul Renuka,
Afzalpurkar Abhijit,
Gupta Satish K.
Publication year - 1997
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/(sici)1098-2795(199706)47:2<140::aid-mrd3>3.0.co;2-m
Subject(s) - microbiology and biotechnology , biology , zona pellucida glycoprotein , antiserum , recombinant dna , zona pellucida , antibody , biochemistry , gene , genetics , embryo , oocyte
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.