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Bovine ephemeral fever (BEF) caused by Bovine ephemeral fever virus (BEFV), is an acute epidemic infection in cattle and water buffalo. The disease was first found in East Africa, and has spread rapidly in many countries of Africa, Asia and Oceania (1, 6, 7). The infection causes considerable economic loss in the cattle industry including the reduced output and quality of the milk, and abortion and lameness or paralysis.
At present, the virus neutralization (VN) test is the standard method for detecting anti-BEFV antibody, however, asepsis is a strict requirement in this method, and consequently it is difficult to perform under the common conditions. In 1992, a blocking enzyme-linked immunosorbent assay (b-ELISA) was estab-lished which could detect specific antibodies to the antigenic site G1 of the BEFV glycoprotein in cattle serum. Compared with the VN test, the b-ELISA was more sensitive and simpler to perform (9). Also, some diagnostic methods based on PCR have been established in some laboratories (2, 5). However, the PCR detecting method has not been used widely. In this paper, the Epitope G1 of BEFV was successfully expressed in P. pastoris GS115 and characterized successfully. These findings provide the basis for the development of an ELISA kit for BEF diagnosis.
Expression and Antigenic Characterization of the Epitope-G1 of the Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris*
- Received Date: 29 November 2006
- Accepted Date: 19 July 2007
Abstract: The epitope-G1 gene of Bovine ephemeral fever virus (BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1. Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS115. The recombinant P. pastoris strains were selected by a G418 transformation screen and confirmed by PCR. After being induced with methanol, an expressed protein with 26 kDa molecular weight was obtained, which was much bigger than the predicted size (15.54 kDa). Deglycosylation analysis indicated the recombinant G1 was glycosylated. Western blot and ELISA tests, as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity. The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.