Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP2.
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 lambda gt11 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 1436 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.
Jethanandani
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Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Santhanam
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Gupta
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SK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004527:Egg Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008255:Macaca radiata; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D011817:Rabbits; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
D018076:DNA, Complementary; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C000606515:ZP2 protein, human; D000072216:Zona Pellucida Glycoproteins