| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 89 === Abstract：
Epstein-Barr virus (EBV) is a member of the herpesviridae. It has been reported that EBV is the etiological agent of infectious mononucleosis and closely associated with African Burkitt’s lymphoma and nasopharyngeal carcinoma (NPC) of southern Chinese.EBV thymidine kinase (TK) is encoded by the BXLF1 ORF and expressed in the early phase of viral lytic cycle. It is the enzyme that phosphorylates thymidine to thymidine monophosphate (TMP) during the salvage pathway of DNA synthesis.
A cDNA of the EBV TK was previously cloned from a cDNA library of P3HR1 cells in this laboratory and designated as TKB1B. The EBV TK coding sequence was subcloned into expression vector pRsetA. It has also been successfully expressed in E. coli system with high quantity and high enzyme activity. The monoclonal antibody 5F4C specific to EBV TK was also obtained before. In this study, the antibody affinity column made of the TK monoclonal antibody was used to purify TK protein. The result shows this method could purify TK protein , and Kms of the proteins before and after purification are almost the same.
Most of the knowledge regarding EBV TK protein structure and reaction mechanism came from the researches on herpes simplex virus (HSV) TK. Although much of EBV TK sequence was similar to HSV TK, these two enzymes are somehow different in biochemical properties and functions. Various herpesviral thymidine kinases were examined and six highly conserved regions were observed by multiple alignment. To study the function of the amino acid residues within the conserved sites, site-directed mutagenesis to generate a series of mutants has been carried out in this laboratory. TK activity assays were performed and Kms of the mutant proteins were determined to map the function of amino acid residues within the putative substrates binding sites of the EBV. The results indicated that amino acids with charge and hydroxyl group in site 1 may be required for ATP binding, and glycine 294 is probable the turn of secondary structure. The amino acids with charge in site 3 may be required for thymidine binding, and histidine 394 plays a crucial role at this position. The specific side chain of residues in site 4 maybe involved in thymidine recognition. These results are compatible with previous studies on TK conserved regions.
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