title: Baculovirus surface display of Theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes.
abstract: Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a severe lymphoproliferative disease in cattle. Previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes have not resulted in a correctly folded protein. Here, we report the expression of N- and C-terminal domains of p67 fused to the baculovirus envelope glycoprotein GP64 by cloning the appropriate p67 cDNA segments between the signal sequence and the major portion of GP64. To further advance the generation of such recombinants, existing surface display techniques were combined with bacmid technology. Chimeric proteins were present on the surface of budded viruses as judged by immunogold labelling and were exposed on the surface of insect cells, as concluded from immunofluorescence studies of infected, non-fixed insect cells. In non-denaturing dot blot experiments, a strong reaction was obtained between monoclonal TpM12 and baculovirus particles displaying the p67N-GP64 chimeric protein. This antibody, raised against native p67, also specifically recognized the surface of recombinant-infected cells. Apparently, a more native conformation was achieved than when p67 was expressed in E.coli or in conventional baculovirus expression systems. The baculovirus surface expression system, therefore, provides an improved way of expressing this T.parva sporozoite surface protein.
authors.affiliation: Laboratory of Virology, Wageningen University, Binnenhaven 11,6709 PD Wageningen, The Netherlands.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Kaba
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Hemmes
authors.forename: Jan W M
authors.initials: JW
authors.lastname: van Lent
authors.forename: Just M
authors.initials: JM
authors.lastname: Vlak
authors.forename: Vishvanath
authors.initials: V
authors.lastname: Nene
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Musoke
authors.forename: Monique M
authors.initials: MM
authors.lastname: van Oers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018411:Spodoptera; D034101:Sporozoites / Q000276:immunology*; D016797:Theileria parva / Q000235:genetics / Q000276:immunology*; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D014760:Viral Fusion Proteins; C114309:p67 antigen, Theileria