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Title: Identification of a 40KD protein increased by HSV-2 infection
Author: Lucasson, Jean-Francois
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Herpes simplex virus (HSV) has been implicated in the etiology of human cancer, but its role in the transformation process is not well understood. A set of cellular polypeptides of 200KD, 90KD (a doublet U90 and L90) and 40KD (TBS:40) was previously detected by immunoprecipitation (i.p.) with tumour bearing serum in the Bn5T cell line. The Bn5T cell line is derived from rat embryo fibroblast transformed by a fragment of the HSV type 2 (HSV-2). These polypeptides were detected in cell lines transformed by other agents and were not detectable in control rat embryo (RE) cells (Macnab et al., 1985). The aim of the project was to purify and obtain amino acid sequence for the TBS:40. The 40KD polypeptide was characterized by its digestion pattern with the enzyme Staph, aureus V8 protease. The U90 and the TBS: 40 were increased upon infection with HSV (Macnab et al., 1992) but this was not known at the start of this thesis. The 40KD protein was purified from Bn5T cells and notfrom infected cells. Attempts to raise antibodies in mice and rabbits were carried out. In rabbit there was no immunological response. To raise monoclonal antibodies (Mab) twelve mice were immunized against Bn5T tumour cells. The 90KD polypeptide i.p. by mouse antisera and the U90 i.p. by TBS were similar, in respect of the Staph, aureus V8 protease peptide map. By contrast the 40KD polypeptide i.p. by the serum of the mice and the TBS:40 had different peptide maps. Therefore, in this instance, the immune system of the rat and the mouse recognized different 40KD proteins. Unfortunately attempts to raise Mabs against the 90KD and the 40KD polypeptides failed. Although the 40KD polypeptide did initially raise Mabs, these were unfortunately subsequently lost. The TBS:40 was purified by biochemical methods. In these experiments TBS was found to i.p. more than one polypeptide. It was decided to purify and identify each polypeptide in turn arid lastly to test the effect of HSV-2 infection on its expression. Ammonium sulphate fractionation separated two 40KD proteins i.p. by TBS. One was mainly insoluble, the other was soluble in a 70% saturated ammonium sulphate solution. The Staph, aureus V8 peptide map of the TBS:40 and the 40KD protein soluble in a 70% saturated ammonium sulphate solution were undistinguishable. Therefore it was decided to purify the soluble 40KD protein. The 40KD protein was further purified by anion exchange chromatography at pH. 8. The 40KD protein eluted in the void volume. The pH. of the void volume was increased to pH.9.5 and anion exchange chromatography at pH. 9.5 separated two 40KD polypeptides, one eluted in the void volume and was called the "VOID VOLUME 40"; the other was eluted from the columm and was called the "COLUMN 40". Both had a Stapli. aureus V8 peptide maps different from the TBS:40 peptide map suggesting that the TBS:40 peptide map was produced when both proteins interact with each other. The "COLUMN 40" was i.p. by TBS but not the "VOID VOLUME 40", these results suggested that the "VOID VOLUME 40" is i.p. as part of a complex. The amino acid sequences obtained from the "VOID VOLUME 40" matched the sequence of the mitochondrial aspartate aminotransferase (mAspAT). The "VOID VOLUME 40" was also immunologically related to the mAspAT in a Western blotting experiment. Expression of polypeptides immunologically related to the mAspAT were increased upon infection with HSV-2. Two peptides obtained by digestion of the "COLUMN 40" were successfully sequenced. The sequence of one matched the sequence of the rat fructose 1-6 diphosphate aldolase, and the sequence of the second peptide matched the sequence of the rat phosphoglycerate kinase-1. The experiment was repeated and the sequence data obtained suggested that the "COLUMN 40" was also related to the mAspAT were obtained in the second experiment. Further experiments must be set up to conform this result.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available