Expression of porcine transmissible gastroenteritis virus genes in E. coli as beta-galactosidase chimaeric proteins.

Abstract

Transmissible gastroenteritis virus (TGEV) causes gastroenteritis in pigs of all ages but has a high mortality in neonatal piglets resulting in severe economical losses in affected pig farms. In piglets, under two weeks of age, the first clinical sign is usually vomiting 18–24h after infection rapidly followed by a diarrhoea, resulting in loss of weight and dehydration; death usually occurs after 2–5 days (Garwes, 1982). The virus is enveloped, contains a single stranded RNA genome of positive polarity and three structural proteins; a 200 000 dalton surface glycoprotein (the spike or peplomer protein), a 30 000 dalton glycoprotein associated with the viral envelope (the integral membrane protein) and a basic 47 000 dalton phosphorylated protein associated with the viral RNA (the nucleoprotein). During replication of the virus in infected cells four subgenomic species of RNA are produced of which three have been shown to produce the three structural proteins (Millson et al. unpublished results). The fourth RNA species does not appear to produce a polypeptide detectable either in infected cells or by in vitro translation of the RNA though it has the capacity to produce a polypeptide of about 33 000 daltons. The nucleoprotein gene has been copied from the smallest subgenomic RNA species, shown by in vitro translation to produce the nucleoprotein (Millson et al. impublished results), into cDNA. The complete DNA sequence of the gene has been determined (Britton et al. unpublished results) and a 1.38kb fragment composed of 245 amino acids, corresponding to 68% of the complete gene product, has been fused to the 3′ end of the E. coli lacz gene. The fused genes produced a β-galactosidase-TGEV nucleoprotein fragment chimaeric protein in E. coli. The chimaeric protein has been purified and used to raise antibodies in mice which immune precipitated only the viral nucleoprotein confirming that the DNA sequence assumed to contain the nucleoprotein gene sequence does so. This method provides a useful source of TGEV antigen for virus diagnostic tests and viral protein for use in a research programme aimed at understanding the mechanisms of antigen processing and immune stimulation. The method is useful for confirming the presence of open reading frames (ORFs) and identifying the true product of genes on cDNA either suspected to contain a particular gene or for those previously identified by DNA sequencing.