目的：制备TAT-ASPP2融合蛋白，探讨其对人胶质瘤U-87MG细胞和U251细胞增殖的抑制作用。方法：设计TAT-ASPP2引物，应用IN-Fusion技术构建原核表达质粒pET-TAT-ASPP2，双酶切、DNA测序鉴定后转化大肠杆菌E.coli BL21，IPTG诱导TAT-ASPP2融合蛋白的表达，SDS-PAGE和Western blotting鉴定TAT-ASPP2融合蛋白。MTT法检测TAT-ASPP2融合蛋白对U-87MG和U251细胞增殖的作用。结果：成功构建了原核表达质粒pET-TAT-ASPP2，转化E.coli BL21后成功表达TAT-ASPP2融合蛋白，其相对分子质量约为128 000，并可被ASPP2特异性抗体所识别。TAT-ASPP2融合蛋白对U-87MG和U251细胞增殖的抑制率分别为(65.0±3.0)%和(64.7±2.5)%，而ASPP2蛋白则不能抑制U-87MG和U251细胞的增殖。结论：成功地克隆、表达及纯化TAT-ASPP2融合蛋白，该融合蛋白可抑制胶质瘤细胞的增殖。

Objective:To prepare the TAT-ASPP2 fusion protein and investigate its inhibitory effect against the proliferation of glioma U-87MG and U251 cells. Methods: TAT-ASPP2 specific primer was designed and recombinant prokaryotic expression vector pET-TAT-ASPP2 was constructed using the In-Fusion cloning technique. After identified by double endonuclease digestion and DNA sequencing, pET-TAT-ASPP2 vector was transformed into E. coli BL21 and TAT-ASPP2 fusion protein was induced by IPTG. TAT-ASPP2 fusion protein was further identified by SDS-PACE and Western blotting analysis. The effects of TAT-ASPP2 fusion protein on proliferation of U-87MG and U251 cells were detected by MTT assay. Results: The prokaryotic expression plasmid pET-TAT-ASPP2 was successfully constructed, and TAT-ASPP2 fusion protein was induced by IPTG in transformed E.coli BL21; the molecular weight of the fusion protein was about 128 000 and it could be specifically recognized by ASPP2 antibody. TAT-ASPP2 fusion protein significantly inhibited the proliferation of U-87MG and U-251 cells, with the inhibitory rates being about (65.0±3.0)% and (64.7±2.5)%, respectively; while ASPP2 protein did not inhibit the proliferation of U-87MG and U-251 cells. Conclusion: TAT-ASPP2 fusion protein has been successfully expressed and purified, and the fusion protein can significantly inhibit the proliferation of glioma cells.

国家自然科学基金资助项目（No. 30800285）；福建省卫生厅青年科研课题（No. 2007-1-20）