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Expression of Porcine Transmissible Gastroenteritis Virus Genes in E. Coli as β-Galactosidase Chimaeric Proteins

  • Paul Britton
  • David J. Garwes
  • Kevin Page
  • Jean Walmsley
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 218)

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 unpublished results).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 unpublished results) and a 1.38kb fragment composed of 245 amino acids, corresponding to 68% of the complete gene product, has been fused to the 3′ end of the E. coli lacz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.

Keywords

lacZ Gene HindIII Site HindIII Fragment Viral Nucleoprotein Transmissible Gastroenteritis Virus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Bankier, A. and Barrell, B.G., 1983, Shotgun DNA sequencing, In: “Techniques in the Life Sciences (Nucleic Acid Biochemistry),” B508: 1. R.A. Flavell, ed., Elsevier, Ireland.Google Scholar
  2. Biggin, M.D., Gibson, T.J., and Hong, G.F., 1983, Buffer gradient gels and S label as an aid to rapid DNA sequence determination, Proc. Nat. Acad. Sci., USA, 80: 3963.CrossRefGoogle Scholar
  3. Britton, P., Murfitt, D., Parra, F., Jones-Mortimer, M.C. and Kornberg, H.L., 1982, Phosphotransferase-mediated regulation of carbohydrate utilisation in Escherichia coli K12: identification of the products of genes on the specialised transducing phages iex(crr) and gsr (tgs), EMBO J., 1: 907.PubMedGoogle Scholar
  4. Chirgwin, J.M., Przybila, A.E., MacDonald, R.S., and Rutter, W.J., 1979, Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18: 5294.PubMedCrossRefGoogle Scholar
  5. Garwes, D.J., 1982, Coronaviruses in Animals, ln: “Virus Infection of the Gastrointestinal Tract,” D.A.J. Tyrrell and A.Z. Kapikian, eds., Marcel Dekker, Inc., New York.Google Scholar
  6. Garwes, D. J., Bountiff, L., Millson, G.C., and Elleman, C.J., 1984, Defective replication of porcine transmissible gastroenteritis virus in a continuous cell line, Adv. Exp. Med. and Biol., 173: 79.Google Scholar
  7. Garwes, D.J., Stewart, F. and Elleman, C.J., 1987, Identification of epitopes of immunological importance on the peplomer of porcine transmissible gastroenteritis virus, In: “Coronaviruses,” M. Lai, ed., Plenum Press, New York.Google Scholar
  8. Kapke, P.A. and Brian, D.A., 1986, Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene, Virology, 151: 41.PubMedCrossRefGoogle Scholar
  9. Messing, J., 1979, A multipurpose cloning system based on the single-stranded DNA bacteriophage M13, Recombinant DNA Technical Bulletin, NIH 2: 43.Google Scholar
  10. Messing, J., 1583, New M13 vectors for cloning, In: “Methods in Enzymology,” 101: 20, R. Wu, L. Grossman and K. Moldave, eds., Academic Press, New York.Google Scholar
  11. Miller, J.H., 1972, “Experiments in Molecular Genetics,” Cold Spring Harbor Laboratory, New York.Google Scholar
  12. Mole, S.E. and Lane, D.P., 1985, Use of simian virus 40 large T-B-galactosidase fusion protein in an immunological analysis of simian virus 40 large T antigen, J. Virol., 54: 703.PubMedGoogle Scholar
  13. Okayama, H., and Berg, P., 1982, High-efficiency cloning of full-length cDNA, Molec. and Cell. Biol., 2: 16.Google Scholar
  14. Pocock, D.H., and Garwes, D.J., 1975, The influence of pH on the growth and stability of transmissible gastroenteritis virus in vitro, Arch. Virol., 49: 239.PubMedCrossRefGoogle Scholar
  15. Ruther, U., and Muller-Hill, B., 1983, Easy identification of cDNA clones, EMBO J., 2: 1791.PubMedGoogle Scholar
  16. Sanger, F., Nicklens, S., and Coulson, A.R., 1977, DNA sequencing with chain terminating inhibitors, Proc. Nat. Acad. Sci., USA, 74: 5463.CrossRefGoogle Scholar
  17. Staden, R., 1982, Automation of the computer handling of gel reading data produced by the shotgun method of sequencing, Nucleic acid Res., 12: 521.CrossRefGoogle Scholar
  18. Steers Jr, E., Cuatrecases, P., and Pollard, H.B., 1971, The purification of ß-galactosidase from Escherichia coli by affinity chromatography, J.Biol. Chem., 246: 196.PubMedGoogle Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Paul Britton
    • 1
  • David J. Garwes
    • 1
  • Kevin Page
    • 1
  • Jean Walmsley
    • 1
  1. 1.AFRC Institute for Animal Disease ResearchCompton, Newbury, BerksUK

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