中文摘要：
　　胸腺核激(Thymidine kinase, TK)是DNA合成的救助途徑中，將胸腺核磷酸化為胸腺核單磷酸的酵素。本實驗室前已將EB病毒TK的cDNA成功轉殖至表現質體pET3cp*，並在大腸菌中成功的表現大量具有活性的EB病毒TK重組蛋白，之後更將TK基因再轉殖至表現質體pET21c，在大腸菌中表現出在羧基端帶有連續組織胺酸殘基序列的TK重組蛋白，並以鎳離子管柱進行了純化。
　　對於EB病毒TK蛋白質結構與反應機制的認識，長久以來來自於對單純疹病毒(herpes simplex virus, HSV)TK蛋白質的研究，雖然兩者的胺基酸序列極為相似，但在生化性質上仍不盡相同。HSV TK蛋白質的Asp162被推測與鎂離子的定位有關。1996年Black等人將其突變為glutamine，結果突變株TK蛋白質的活性大幅下降，對鎂離子的親和力卻沒有明顯改變。為了證明EB病毒TK蛋白質上的相對位置，Asp392，是否具有相似的功能，本實驗將其亦突變為glutamine，並利用鎳離子管柱純化突變株TK蛋白質後分析其特性。結果與HSV TK蛋白質的研究相似，活性明顯下降，但與鎂離子的結合能力卻與HSV TK蛋白質的研究結果不同，突變株EB病毒TK蛋白質的活性似乎受到鎂離子的抑制。
　　藉由冷凍-解凍再過濾的過程，發現了藉由pET21c表現載體所表現的TK重組蛋白質有強烈的凝聚現象，利用pET3cp*表現的TK重組蛋白質則無此現象，推測可能是TK重組蛋白末端的連續組織胺酸殘基序列造成了較容易凝聚現象。
鼻咽癌是台灣十大癌症死因之一。而抗EB病毒DNA分解抗體是一項檢測鼻咽癌的有效指標。由於鼻咽癌的發生與遺傳及環境因素有關，本實驗篩檢了51個含有鼻咽癌病患的家族（包含了49名病患與257名家屬），141名電信局員工與81名報社員工。近來發現胃癌的發生也與感染EB病毒有關，因此本實驗也針對213名胃癌病患與84名非胃癌病患進行篩檢。
實驗結果顯示鼻咽癌病患家屬的抗EB病毒DNA分解抗體陽性率是9.7%，將近正常人 (約5%) 的兩倍。而針對鼻咽癌病患所測得的陽性率 (65.3%) 雖然低於以往實驗室所測得的值 (80~90%)，但是其中大多數呈現陽性的患者具有高效價的抗EB病毒DNA分解抗體。檢測電信局員工的結果，其陽性率(5.6%) 與正常人並無明顯差異。但在檢測報社員工的結果中卻發現了明顯偏高的陽性率 (25.9%)。針對胃癌病患所測得的陽性率 (16.9%) 與本實驗室已往檢測其他種癌症病患的陽性率 (10~30%) 近似，但在檢測非胃癌患者時卻發現了不尋常的高陽性率，推測可能是由於實驗誤差所導致的假陽性率。

Abstract:
Thymidine kinase (TK) is the enzyme that phosphorylates thymidine (TdR) to thymidine monophosphate (TMP) during the salvage pathway of DNA synthesis. In this laboratory, a recombinant Epstein-Barr virus (EBV) TK cDNA, pET3cp*-TKB1B, has been previously constructed. The recombinant TK protein, 3cp*-TK, has also been successfully expressed in E. coli system with high quantity and high enzyme activity. The EBV TK coding sequence was also subcloned into pET21c and another recombinant EBV TK protein, 21c-TK, with 6 histidine residues at the carboxyl terminus was obtained, allowing the purification of TK protein through a nickel affinity column.
The knowledge of EBV TK protein structure and its reaction mechanism mostly came from researchs on herpes simplex virus (HSV) TK. Though sharing lots of sequence similarities, these two enzymes are still somehow different in biochemical properties. Asp162 of HSV TK has been suggested to function for coordinating Mg2+. To find out if the analog of Asp162 in EBV TK, Asp392, plays the same role, an EBV TK mutant with a glutamine substituted Asp392 was generated and named 21c-TK(D392Q). Both 21c-TK(D392Q) and 21c-TK were purified via nickel affinity column. As a result, 21c-TK(D392Q) exhibited similar properties as the HSV TK D162Q mutant protein except that its activity seemed to be inhibited by Mg2+ .
Through the freezing-thawing process, purified 21c-TK and 21c-TK(D392Q) aggregated seriously. To discover the cause of aggregation, the tendency to aggregation for TK proteins was compared between 21c-TK and 3cp*-TK. After
35,000 rpm centrifugation for 1 hour and filtration, 21c-TK still aggregated much more obviously than 3cp*-TK did. The results suggested that the extra His-tag at the carboxyl terminus of 21c-TK might contribute the easier aggregation.
Nasopharyngeal carcinoma (NPC) is one of the ten most common cancers in Taiwan. Antibody to EBV specific DNase has been proved to be a useful marker for early detection of NPC. NPC development is affected by genetic factors and environmental factors. Therefore, family members from 51 NPC patients (including 49 patients and 257 relatives), 141 employees of a telegraph office and 81 employees of a newspaper company were examined. Gastric cancer is lately proved associated with EBV infection. Thus, sera from 213 gastric carcinoma patients and 84 patients without gastric carcinoma were also included.
The result shows the anti-EBV DNase positive rate of NPC patients’ families is nearly twice the value of healthy group examined previously (9.7% vs 5%). The positive rate of NPC patients is lower than previous results (65.3% vs 80~90%), yet most of the positive individuals have high level anti-EBV DNase. The positive rate of employees of a telegraph office shows no difference with healthy group positive rate (5.6% vs 5%), whereas a significant high positive rate (25.9%) was detected in employees of a newspaper company. Gastric carcinoma patients examined have similar positive rate with other cancer patients examined previously (16.4% vs 10~30%). The non-gastric carcinoma patients have an unusual high positive rate (50%), possibly due to examination error.

目 錄
中文摘要………………………………………. I
英文摘要……………………………………… II
縮寫表………………………………………. III
序論……………………………………………. 1
實驗材料與方法…………………………….. 18
實驗結果…………………………………….. 35
討論………………………………………….. 43
圖表………………………………………….. 51
實驗步驟…………………………………….. 76
參考文獻…………………………………….. 96

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