Expression of env gene of bovine leukemia virus in rodent cells

Zajac, Vladimír; Sláviková, K; Babušíková, O

doi:10.1007/bf01309779

Cited by 1 publication

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“…2). All the proteins that migrated faster than native gp51 could represent incomplete and/or incorrectly glycosylated molecules, as is frequently observed in insect cells (16,19) and different mammalian cell lines (1,26).…”

Section: Discussionmentioning

confidence: 89%

Expression of the Bovine Leukemia Virus Envelope Glycoprotein (gp51) by Recombinant Baculovirus and Its Use in an Enzyme-Linked Immunosorbent Assay

Giuseppe

Feliziani

Rutili

et al. 2004

Clin Vaccine Immunol

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The gene encoding the major envelope glycoprotein (gp51) with its signal sequence, represented by an additional NH 2 -terminal 33-residue amino acid sequence of bovine leukemia virus (BLV), was inserted into a baculovirus transfer vector. A recombinant virus expressing a secreted gp51 protein in insect cells was isolated. The recombinant gp51 expressed was characterized by using an anti-BLV monoclonal antibody by both Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). The secreted gp51 was used as an antigen, and an ELISA with recombinant gp51 (rgp51) was developed for the detection of BLV antibodies. This new procedure was compared with a previous ELISA method for the detection of BLV antibodies and an agar gel immunodiffusion test performed with an unpurified BLV antigen preparation. The comparative testing of field samples showed that the ELISA with rgp51 is more specific and also suitable for the testing of pooled sera.Bovine leukemia virus (BLV), the etiological agent of enzootic bovine leukosis (EBL), is a C-type retrovirus of cattle with a worldwide distribution (5). The virus induces a persistent lymphocytosis and in some cases lymphoid tumors in domestic cattle.The BLV envelope (Env) glycoprotein, which consists of the gp51 outer membrane glycoprotein and the gp30 transmembrane glycoprotein, is directly involved in infectivity events and, like the p24 major structural protein, can elicit a strong immune response in infected cattle (23). Serological diagnosis of EBL is mainly based on screening of field samples for gp51 antibodies. It follows that diagnostic procedures that use native gp51 constitute a prerequisite for the design of an efficient EBL eradication program. In recent decades different serological methods, such as the agar gel immunodiffusion test (AGID) and the enzyme-linked immunosorbent assay (ELISA), have been developed. ELISAs are based on the use of partially purified BLV gp51 and monoclonal antibodies (MAbs) against BLV gp51 epitopes (4, 17). These procedures are particularly useful for samples with low antibody titers, such as milk samples or pooled sera. A permanent fetal lamb kidney (FLK) cell line chronically infected with BLV (FLK-BLV) is used to produce gp51. Although highly productive BLV-infected cell lines are available, their production is rather laborious, expensive, and time-consuming.In recent decades different investigators have described the expression of the BLV Env glycoproteins in heterologous expression systems such as Escherichia coli (2, 21, 22), Saccharomyces cerevisiae (11), and recombinant vaccinia virus (10, 18) systems and, recently, a baculovirus system (9, 19). However, the use of recombinant gp51 (rgp51) in an ELISA method has not yet been described. The aim of this paper is therefore to describe the production of secreted rgp51 by recombinant baculovirus in insect cells and its use in an ELISA for the detection of BLV antibodies. MATERIALS AND METHODS Cells and viruses.BLV-infected and uninfected FLK cells (National Veterinary Laborat...

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