Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies

Abstract

Pesq. Vet. Bras. 33(1): 41-46, janeiro 2013 41 RESUMO.-[Expressão procariota de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 e uso em ELISA para anticorpos contra a gE.] Este trabalho relata a expressão de uma forma truncada da glicoproteína E (gE) do herpesvírus bovino tipo 1 (BoHV-1) para uso em imunodiagnóstico. Um fragmento de 651 pares de bases (pb) correspondente ao terço amino-terminal (217 aminoácidos) da gE do BoHV-1 -que compartilha uma alta identidade com a gE do BoHV-5 -foi clonada como proteína de fusão com cauda 6x de histidina em um vetor de expres- This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE -that shares a high identity with the homologous BoHV-5 counterpart -was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines. bovine herpesvirus, vaccine, DIVA, recombinant protein. são em Escherichia coli. Uma proteína solúvel de aproximadamente 25 kDa purificada de lisados de E.coli foi reconhecida em Western blot (WB) por anticorpos monoclonais anti-6xHis-tag e anti-gE. Além disso, a proteína recombinante purificada foi reconhecida em WB por anticorpos presentes no soro de animais soropositivos ao BoHV-1 e BoHV-5. Um ELISA indireto utilizando a proteína recombinante como antígeno apresentou performance comparável a um ELISA gE comercial e foi capaz e diferenciar sorologicamente animais vacinados com uma cepa gE-negativa de BoHV-5 de animais infectados com o BoHV-1. Portanto, a gE truncada pode ser útil em testes sorológicos diferenciais para uso conjunto com vacinas com marcador antigênico gE para o BoHV-1 e BoHV-5.TERMOS DE INDEXAÇÃO: BoHV-5, herpesvírus bovino, vacina diferencial, proteína recombinante.

Serological Evidence of Hepatitis E Virus Infection in Brazilian Equines

“…Serum samples obtained from colostrum-deprived newborn foals (n = 18) were used as negative controls (a strategy also employed for donkeys [22]) since there is no transference of immunoglobulins through the diffuse epitheliochorial placenta of the mares [33]. However, when applied to the formula cutoff = mean of negative controls + 3 × SD [39][40][41], the mean optical density (OD 0.0263 ± 0.006) obtained for the foal's sera resulted in an overestimated anti-HEV IgG detection rate of more than 99% positivity (up to 18% in previous studies) [22,23,34,35]). The challenge of setting a cutoff value without known negative and positive samples could be overcome by applying the changing-point statistical algorithm, which does not require predefined samples for calculation.…”

Secretory expression of bovine herpesvirus type 1/5 glycoprotein E in Pichia pastoris for the differential diagnosis of vaccinated or infected cattle

“…Truncated forms of gE from BoHV-1 have been expressed successfully using Baculovirus-insect systems [34e36], mammalian cells [37] and prokaryotic expression methods [35,38]. However, as gE contains several antigenic domains, detection of anti-gE antibodies is likely to be enhanced using the whole protein [39,40].…”