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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.coliBL21,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-AS-PP2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128000,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 TAT-ASPP2 fusion protein and investigate its inhibitory effect on the proliferation of U-87MG cells and U251 cells. Methods: The TAT-ASPP2 primer was designed. The prokaryotic expression plasmid pET-TAT-ASPP2 was constructed by IN-Fusion technique. The recombinant plasmid was identified by DNA sequencing and transformed into E.coli BL21. IPTG induced the expression of TAT-ASPP2 fusion protein. SDS- PAGE and Western blotting to identify TAT-ASPP2 fusion protein. MTT assay TAT-AS-PP2 fusion protein on U-87MG and U251 cell proliferation. Results: The prokaryotic expression plasmid pET-TAT-ASPP2 was successfully constructed. After transformed into E. coli BL21, the TAT-ASPP2 fusion protein was successfully expressed, with a relative molecular mass of about 128,000 and was recognized by ASPP2-specific antibodies. The inhibitory rates of TAT-ASPP2 fusion protein on proliferation of U-87MG and U251 cells were (65.0 ± 3.0)% and (64.7 ± 2.5)% respectively, while ASPP2 protein could not inhibit the proliferation of U-87MG and U251 cells. Conclusion: TAT-ASPP2 fusion protein was successfully cloned, expressed and purified. This fusion protein can inhibit the proliferation of glioma cells.