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目的:制备重组hS100A6蛋白,并研究其对人骨肉瘤细胞系143B的生物学作用。方法:构建pGST-HRV3C-hS100A6质粒,经转化至E.coli BL21,IPTG诱导表达融合蛋白GST-HRV3C-hS100A6,经超声破菌,谷胱甘肽-琼脂糖4B球珠纯化,GST-HRV3C酶切,再纯化,Western blot鉴定,分光光度法蛋白定量。以人骨肉瘤细胞系143B为研究对象,MTT检测细胞增殖,Hoechst检测细胞凋亡,Transwell检测细胞迁移和侵袭,Western blot检测hS100A6对β-catenin表达的影响。结果:成功制备重组hS100A6蛋白,测得该蛋白产量约为4mg/L菌液。30μg/ml重组hS100A6促进143B细胞的增殖、迁移、侵袭以及β-catenin的表达(P<0.05),对143B细胞的凋亡无明显影响(P>0.05)。结论:成功制备重组hS100A6蛋白,30μg/ml重组hS100A6蛋白对143B细胞有一定的促进作用。
OBJECTIVE: To prepare recombinant hS100A6 protein and study its biological effect on human osteosarcoma cell line 143B. Methods: The pGST-HRV3C-hS100A6 plasmid was constructed and transformed into E.coli BL21. The fusion protein GST-HRV3C-hS100A6 was induced by IPTG. After being purified by ultrasonication and glutathione-Sepharose 4B, GST-HRV3C Cut, re-purified, Western blot identification, spectrophotometric protein quantification. The human osteosarcoma cell line 143B was used as the research object. Cell proliferation was detected by MTT assay. Cell apoptosis was detected by Hoechst assay. Transwell assay was used to detect cell migration and invasion. Western blot was used to detect the effect of hS100A6 on β-catenin expression. Results: The recombinant hS100A6 protein was successfully prepared and its yield was about 4 mg / L. The proliferation, migration, invasion and β-catenin expression of 143B cells were significantly inhibited by 30μg / ml recombinant hS100A6 (P <0.05), but had no significant effect on the apoptosis of 143B cells (P> 0.05). Conclusion: The successful preparation of recombinant hS100A6 protein, 30μg / ml recombinant hS100A6 protein 143B cells have a certain role in promoting.