Department of Microbiology and Parasitology, Shanghai Medical College of Fudan University, No. 138 Yixueyuan Road, Shanghai 200032, China.
Parasitol Res. 2012 Feb;110(2):931-7. doi: 10.1007/s00436-011-2579-x. Epub 2011 Aug 14.
Apyrases (ATP diphosphohydrolase) hydrolyze the phosphodiester bonds of nucleoside tri- and diphosphates to orthophosphate and mononucleodides. They can inhibit platelet activation by depletion of adenosine diphosphate. In the current study, the Escherichia coli expression vector pET-19b equipped with an N-terminal histidine tag was applied to express the apyrase of Aedes albopictus. The gene-coding mature apyrase protein was amplified by RT-PCR and cloned into pET-19b. Soluble apyrase protein with high purity was successfully obtained by utilization of the suitable renaturation approach and Ni-NTA purification column. Four monoclonal antibodies to apyrase from A. albopictus were produced in male BALB/c mice immunized with the renatured apyrase. Using immunofluorescence assay and immunoblotting analysis, recombinant apyrase showed fine consistency with native apyrase. From kinetic analysis, it had a K (m) of 11.6 μM and V (max) of 1.02 nM/S/μg protein for adenosine triphosphate. Adenosine diphosphate-induced platelet aggregation was inhibited by approximately 6% when 0.4 μM recombinant apyrase was added and by about 9.5% when the concentration of recombinant apyrase was 0.8 μM. The effect on platelet aggregation was dose dependent. In conclusion, the apyrase of A. albopictus was cloned and expressed highly in the E. coli expression system. Recombinant apyrase protein showed biological activity, and anti-apyrase monoclonal antibody was also prepared.
磷酸酶（ATP 二磷酸水解酶）可将核苷三磷酸和二磷酸的磷酸二酯键水解为正磷酸盐和单核苷酸。它们可以通过耗尽二磷酸腺苷来抑制血小板活化。在本研究中，使用带有 N 端组氨酸标签的大肠杆菌表达载体 pET-19b 表达白纹伊蚊的磷酸酶。通过 RT-PCR 扩增编码成熟磷酸酶蛋白的基因，并将其克隆到 pET-19b 中。通过利用合适的复性方法和 Ni-NTA 纯化柱，成功获得了高纯度的可溶性磷酸酶蛋白。用复性磷酸酶免疫雄性 BALB/c 小鼠，产生了针对白纹伊蚊磷酸酶的 4 种单克隆抗体。通过免疫荧光和免疫印迹分析，重组磷酸酶与天然磷酸酶具有很好的一致性。从动力学分析，它对三磷酸腺苷的 K（m）为 11.6 μM，V（max）为 1.02 nM/s/μg 蛋白。当加入 0.4 μM 重组磷酸酶时，可抑制约 6%的二磷酸腺苷诱导的血小板聚集，当重组磷酸酶浓度为 0.8 μM 时，可抑制约 9.5%的血小板聚集。该抑制作用呈剂量依赖性。总之，白纹伊蚊的磷酸酶被克隆并在大肠杆菌表达系统中高效表达。重组磷酸酶蛋白表现出生物活性，并且还制备了抗磷酸酶单克隆抗体。
