Document 0350
 DOCN  M94A0350
 TI    Expression and purification of nonglycosylated SIV proteins, and their
       use in induction and detection of SIV-specific immune responses.
 DT    9412
 AU    Hanke T; Botting C; Green EA; Szawlowski PW; Rud E; Randall RE; School
       of Biological and Medical Sciences, Division of Cell and; Molecular
       Biology, University of St. Andrews, Fife, U.K.
 SO    AIDS Res Hum Retroviruses. 1994 Jun;10(6):665-74. Unique Identifier :
       AIDSLINE MED/94355111
 AB    Two commercially available expression vectors were modified to generate
       plasmids pGEXcPk and pQ9cPk. Proteins expressed from pGEXcPk and pQ9cPk
       had a short oligopeptide tag termed Pk at their carboxy termini and
       either glutathione S-transferase (GST) or a small histidine (His) tag,
       respectively, at their N termini. GST fusion proteins can be purified on
       immobilized glutathione and proteins coupled to the His tag selectively
       bind to Ni(2+)-NTA columns. The Pk tag is recognized by monoclonal
       antibody (MAb) SV5-P-k, previously produced in our laboratory. Thus
       proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified
       in a two-step procedure, first via the N-terminal tag and second via the
       C-terminal tag. The combination of two affinity purification steps
       significantly improves the antigen purity and selects for full-size
       proteins. Moreover, by using the MAbSV5-P-k in the second purification
       step, Pk-linked antigens can be assembled directly into solid
       matrix-antibody-antigen (SMAA) complexes for use as vaccines. The genes
       for nef, endonuclease, p15, p17, p27, protease, Rev, reverse
       transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency
       virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and
       His-SIV-Pk proteins. Multivalent SMAA complexes were made that contained
       His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk. Following
       two immunizations of mice with this mixture, antibodies could be
       detected to all five SIV antigens. When compared to single-protein
       immunizations, the immunogenicity of some of the proteins in this
       cocktail was either enhanced or decreased. Mice were also immunized with
       His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence
       of alum. The antibody-antigen complexes induced two- to four-fold higher
       antibody levels than antigen alone but did not appear to be more
       immunogenic in inducing lymphoproliferative responses. Sera from
       SIV-infected macaques were tested for the presence of antibodies
       reacting with the recombinant proteins by Western blot analysis.
       Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily
       detected, antibodies against protease and vpx were present at much lower
       levels, but no antibodies were detected to nef, rev, tat, or vpr. Thus,
       we have developed a comprehensive range of reagents (available on
       request) that can be used to examine immune responses to SIV in both
       mice and monkeys.
 DE    Animal  Antigen-Antibody Complex/IMMUNOLOGY  Escherichia coli/GENETICS
       Macaca  Mice  Plasmids  Support, Non-U.S. Gov't
       SIV/*CHEMISTRY/*IMMUNOLOGY  Viral Fusion Proteins  Viral
       Proteins/GENETICS/*IMMUNOLOGY/*ISOLATION & PURIF  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).