title: Cloning and expression of an antigenic domain of glycoprotein gE of pseudorabies virus in Escherichia coli and its use as antigen in diagnostic assays.
abstract: Use of a combination of an effective gE gene-deleted pseudorabies virus (PRV) vaccine with a companion diagnostic kit for PRV glycoprotein gE has proven successful in several pseudorabies-eradication programs. To produce a large quantity of functional gE protein for development of a PRV-gE diagnostic kit, an Escherichia coli expression system containing the distal region of the PRV-gE gene of a PRV strain CF was constructed. The expressed protein contained 134 amino acids of gE protein (amino acids 77-210) fused to a 19-amino acids tag containing 6 histidine residues. After induction, a truncated PRV-gE polypeptide of 18-kd was expressed to about 20% of the total E coli proteins. Results of immunoblot analysis indicated that this E coli-produced PRV-gE protein reacted specifically with serum from PRV-hyperimmunized pigs and from field PRV-infected pigs, but not with serum samples from specific-pathogen-free pigs or pigs inoculated with gE-deleted PRV vaccine. These data indicate that, although the recombinant gE protein is produced in E coli, it still retains the antigenicity of the viral gE glycoprotein. Comparison between the recombinant gE protein, using immunoblot analysis with a commercial gE ELISA containing natural PRV-gE protein, revealed comparable test performance. This finding indicated that recombinant gE protein produced by E coli can be used for development of a companion serologic assay for a PRV-gE gene-deleted vaccine.
authors.affiliation: Molecular Biology Division, Development Center for Biotechnology, Taipei, Taiwan, Republic of China.
authors.forename: L H
authors.initials: LH
authors.lastname: Ro
authors.forename: S S
authors.initials: SS
authors.lastname: Lai
authors.forename: W L
authors.initials: WL
authors.lastname: Hwang
authors.forename: H H
authors.initials: HH
authors.lastname: Chou
authors.forename: J N
authors.initials: JN
authors.lastname: Huang
authors.forename: E L
authors.initials: EL
authors.lastname: Chang
authors.forename: H L
authors.initials: HL
authors.lastname: Yang
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015139:Blotting, Southern / Q000662:veterinary; D003001:Cloning, Molecular / Q000379:methods; D015342:DNA Probes; D004926:Escherichia coli; D011558:Herpesvirus 1, Suid / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010957:Plasmids; D011557:Pseudorabies / Q000175:diagnosis* / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012698:Serologic Tests; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D015342:DNA Probes; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C089580:glycoprotein E, Suid herpesvirus 1