title: The production and purification of PCR-derived recombinant simian immunodeficiency virus p27 gag protein; its use in detecting serological and T-cell responses in macaques.
abstract: The polymerase chain reaction (PCR) was used to amplify a region of the gag gene, encompassing the core protein p27, from genomic DNA of cells infected with SIVmac251 (32H isolate). The 767 base pair PCR product was cloned into the bacteriophage M13 and fully sequenced before sub-cloning into the expression vector pUC19. The 30 kilodalton (kDa) fusion protein of lacZ-p27 was expressed as a soluble protein in E. coli JM101 cells and purified to greater than 90% purity by affinity chromatography. The affinity purified product was used in serological and T-cell assays to assess immune function in cynomolgus macaques immunised or challenged with immunodeficiency virus derived material. This reagent and accompanying methods provide valuable assays for monitoring the efficacy of vaccines for SIV as a model for human AIDS.
authors.affiliation: Collaborative Centre for AIDS, National Institute for Biological Standards and Control, South Mimms, Potters Bar, U.K.
authors.forename: N
authors.initials: N
authors.lastname: Almond
authors.forename: M
authors.initials: M
authors.lastname: Page
authors.forename: K
authors.initials: K
authors.lastname: Mills
authors.forename: A
authors.initials: A
authors.lastname: Jenkins
authors.forename: C
authors.initials: C
authors.lastname: Ling
authors.forename: R
authors.initials: R
authors.lastname: Thorpe
authors.forename: P
authors.initials: P
authors.lastname: Kitchin
authors.forename: M
authors.initials: M
authors.lastname: Williams
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics* / Q000276:immunology; D014644:Genetic Variation; D007763:Lac Operon; D008213:Lymphocyte Activation / Q000276:immunology; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012192:Retroviridae Infections / Q000235:genetics / Q000276:immunology; D015302:Simian Immunodeficiency Virus / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; C066594:Gag protein p27, Simian immunodeficiency virus; D015683:Gene Products, gag; D011994:Recombinant Proteins