本實驗為研究絲瓜簇葉病植物菌質體tmk 基因段。以引子tmk-1與 tmk-2，利用聚合?連鎖反應(PCR) 擴增絲瓜簇葉病植物菌質體tmk 基因段，再利用EcoR1 與 BamH1 限制?切位將此基因段構築至pGEX-2T 蛋白質表現載體之中。將此重組的載體表現在大腸桿菌XL1-blue寄主細胞中大量表現並且在IPTG誘導之下生合成由GST與胸?酸激?(TMK)組成之融合蛋白。此蛋白質為分子量大約50 kD， 在應用親和性Glutathione Sepharose 4B管柱萃取純化以及thrombin酵解之後便得到24 kD左右的胸?酸激?蛋白質。 此胸?酸激?催化磷酸根基團的轉移從ATP轉給dTMP。連續的分光儀分析加上運用丙酮酸磷酸?與乳酸去氫?做偶合反應，用來分析胸?酸激?酵素活性反應以及酵素動力學研究。在酵素活性反應中還原態的NADH被氧化並且在波長340 nm 吸光值發生變化，我們便可以觀察到胸?酸激?的活化催化作用。其反應pH 值約為pH 7 而鎂離子(Mg++)濃度為1.5 ∼ 2.0 mM 而最佳溫度為30 ℃。酵素胸?酸激?的km 與Vmax 值為 0.058 mM 和 1.967 μmol/min 。 The tmk gene fragments were amplified on the DNA of phytoplasma associated with loofah witches' broom by polymerase chain reaction (PCR) using the two primers, tmk-1 and tmk-2, and constructed into the pGEX-2T expression vector at EcoR1 and BamH1 sites. The recombinant vector was expressed in E. coli XL1-blue and a fusion protein composed of glutathione S-transferase (GST) and thymidylate kinase (TMK) was produced in the presence of IPTG. The protein had a molecular weight of about 50 kD. After purified by Glutathione Sepharose 4B column and treated by thrombin, a TMK protein of about 24 kD was obtained. The enzyme TMK catalyzes the transfer of phosphate group from ATP to thymidine monophosphate. A continuous spectrophotometric assay using the pyruvate kinase (PK) and lactate dehydrogenase (LDH) coupling system was employed for the TMK activity assay. The reduced-form NADH was oxidized in the reaction and the absobance at 340 nm was measured. The optimum pH for the reaction was about 7 and Mg++ concentration was 1.5 ∼ 2.0 mM at 30 ℃. The km and Vmax of enzyme TMK was 0.058 mM and 1.967 μmol/min, respectively.
