Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1998-5-14
pubmed:abstractText
An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplified SacI and PstI restricted fragment was cloned in-frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein in Escherichia coli. Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-beta-D-thiogalactopyronoside. Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3beta-a homologue of DZP3 and cross-reactive with dog zona pellucida), revealed a major band of 42 kDa. Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction. Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture. Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with porcine ZP3beta but failed to react with porcine ZP3alpha in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with zona pellucida. The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1046-5928
pubmed:author
pubmed:copyrightInfo
Copyright 1998 Academic Press.
pubmed:issnType
Print
pubmed:volume
12
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
331-9
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:9535700-Animals, pubmed-meshheading:9535700-Base Sequence, pubmed-meshheading:9535700-DNA Primers, pubmed-meshheading:9535700-Dogs, pubmed-meshheading:9535700-Egg Proteins, pubmed-meshheading:9535700-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:9535700-Enzyme-Linked Immunosorbent Assay, pubmed-meshheading:9535700-Escherichia coli, pubmed-meshheading:9535700-Female, pubmed-meshheading:9535700-Fermentation, pubmed-meshheading:9535700-Gene Expression Regulation, Developmental, pubmed-meshheading:9535700-Immune Sera, pubmed-meshheading:9535700-Immunoblotting, pubmed-meshheading:9535700-Kinetics, pubmed-meshheading:9535700-Male, pubmed-meshheading:9535700-Membrane Glycoproteins, pubmed-meshheading:9535700-Polymerase Chain Reaction, pubmed-meshheading:9535700-Rabbits, pubmed-meshheading:9535700-Receptors, Cell Surface, pubmed-meshheading:9535700-Recombinant Proteins, pubmed-meshheading:9535700-Time Factors, pubmed-meshheading:9535700-Zona Pellucida
pubmed:year
1998
pubmed:articleTitle
Dog zona pellucida glycoprotein-3 (ZP3): expression in Escherichia coli and immunological characterization.
pubmed:affiliation
Gamete Antigen Laboratory, Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110 067, India.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't