Expression of genes encoding two major Theileria annulata merozoite surface antigens in Escherichia coli and a Salmonella typhimurium aroA vaccine strain.
The genes, Tams1-1 and Tams1-2, encoding the 30-and 32-kDa major merozoite surface antigens of Theileria annulata (Ta), have recently been cloned and characterized. Both genes encode a protein of 281 amino acids (aa) containing a putative hydrophobic N-terminal signal peptide. Another hydrophobic stretch is predicted at the C terminus which probably functions to anchor the protein in the membrane of the merozoite and piroplasm. Here, we report the successful expression of both Tams1-1 and Tams1-2 in Escherichia coli (Ec) using gene fragments lacking both hydrophobic domains. Attempts to produce high amounts of the entire recombinant (re-) protein, or a fragment containing the N terminus only, were unsuccessful. This is presumably due to the toxicity of these re-proteins. The internal part of both genes was also expressed in Salmonella typhimurium (St) aroA vaccine strain SL3261. We employed a dual-plasmid expression system based on an invertible promoter and selected the most stable St construct in vitro using liquid cultures and a macrophage-like cell line. The re-Tams1-1 protein produced in Ec, as well as in St, was recognized by monoclonal antibody (mAb) 5E1 specific to the 30-kDa protein. Both re-Tams1-1 and re-Tams1-2 were recognized by Ta immune calf serum.
d'Oliveira
C
C
Department of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
Tijhaar
E J
EJ
Shiels
B R
BR
van der Weide
M
M
Jongejan
F
F
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D000954:Antigens, Surface / Q000235:genetics*; D001428:Bacterial Vaccines / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D012486:Salmonella typhimurium / Q000235:genetics; D016796:Theileria annulata / Q000235:genetics* / Q000276:immunology
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D001428:Bacterial Vaccines; D017931:DNA Primers