ⅠAnalysis of Epstein-Barr Virus DNA Nucleotide Sequence in Patients with nasopharyngeal Carcinoma

• Main Author: 甘士欣
• Other Authors: 陳振陽
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/34195157291129755776

碩士 === 國立臺灣大學 === 微生物學研究所 === 85 === Nasopharyngeal carcinoma (NPC) is one of the common human malignancies in southern China and Taiwan. It is known that the Epstein-Barr virus (EBV) is one of the contributing factors in NPC. Most NPC is latently infected and expresses a limited number of viral genes, including EBNA-1, LMP1 and LMP2A/2B predominantly. Previous studies have identified sequence alterations relative to the EBV B95-8 strain in the EBNA-1, LMP1, LMP2A/2B genes in different EBV-associated diseases. To understand the possible role of EBV to NPC, we used 14 purified DNA from NPC biopcies, 4 EBV positive cell lines, and 5 lymphoblastoid cell lines (LCL) from Australia as template. Polymerase chain reaction (PCR) was used to amplify partial sequence of LMP1, LMP2A/2B and EBNA-1 genes. DNA sequences were determined by PCR direct sequencing. The result showed that the nucleotide sequences of the PCR products from the NPC specimens actually contained variations relative to B95-8 strain. But the relationship between these polymorphism and protein function is still unclear. And it might confer a character of Taiwan strain. Thymidine kinase (TK) is responsible for phosphorylating thymidine to the thymidine monophosphate in the salvage pathway of DNA synthesis. We have subcloned pTKB1B, a cDNA clone of EBV TK from a cDNA library of P3HR1, into a pET32a+ vector, and also subcloned a mutated TK protein lacking the 240 amino acids at the amino terminus. The recombinant plasmids were transformed into the E. coli BL21 (DE3) strain. The enzymatically active recombinant fusion protein was expressed abundantly by adding IPTG for induction. We found that the truncation of amino terminus the EBV TK will not affect the activity of thymidine phosphorylation. On the other hand the EBV TK was purified by using the nickel affinity resin, only very low yield of purified products could be obtained because of the low binding affinity. We estimate the competition ability to thymidine by adding different nucleosides or nucleoside analogs into EBV TK enzyme mixture. We found that only BrdU and lUdR could compete thymidine in TK assay predominantly, while other nucleosides and nucleoside analogs could not. However, we found that the synthetic drugs had partially competition ability to thymidine in TK assay.