engleski

Identification of glycoproteins gE and gC of the pseudorabies virus strain B-Kal 68 by SDS-PAGE and monoclonal antibodies

Background and purpose: Pseudorabies virus (PRV) is an important pathogen in pig. Some attenuated strains, obtained by multiple passages in chicken embryo fibroblasts (like B-Kal 68), are known to contain genomic deletions that invariably comprise the gene coding for gE. In this study monoclonal antibodies (MoAbs) against gE and gC of highly virulent PRV strain NIA-3 have been produced. The presence of these glycoproteins in PRV B-Kal 68 vaccine strain has been confirmed. Materials and methods: The virus envelope of PRV NIA-3 strain was removed with Triton X-100 and layered onto sucrose gradient. The position of glycoproteins gE and gC in fractions was determined by SDS-PAGE. MoAbs have been produced applying mouse immunization shedule with fractions containing gE and gC. The resulting hybridomas were screened for immunoglobulin production by ELISA using polystirene microtitre plates coated by PRV NIA-3 strain. Protein specificities of prepared MoAbs were carried out by Western blot. The virus envelope of attenuated B-Kal 68 strain was analysed in the same manner and the presence of gE and gC in fractions was analysed by ELISA. Results: It was shown by SDS-PAGE that the largest proteins in fractions 17 through 21 were proteins with Mm of approximately 120 kD (gE) and 92 kD (gC). The presence of gB complex on the opposite side of the gradient was confirmed by ELISA using previously prepared MoAb SHV125C7. Two MoAbs were obtained: MoAb E58H12 reacted with gE (130 kD) ; MoAb E434F4 recognized the mature 92 kD form of gC and its 74 kD precursor. SDS-PAGE protein profile as well as binding of MoAbs in ELISA, where fractions from sucrose gradient of PRV-B-Kal 68 strain were used as antigen, showed that this strain contains fully processed gE and gC in the virus envelope. Conclusions: Our findings is that gE of B-Kal 68 strain is indistinguishable, by its electrophoretic behaviour and recognition by specific MoAb, from that of NIA-3 strain. Together with the fact that B-Kal 68 strain possess thymidine kinase positive phenotype, it indicates that attenuation of this strain is the consequence of so far unknown mutation/s and/or deletion/s.

Pseudorabies virus ; glycoprotein E ; glycoprotein C ; monoclonal antibodies ; SDS-PAGE

nije evidentirano