摘要
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 preparation recombinant hS100A6 protein and study its biological effects on human osteosarcoma cell line 143B. Methods： Recombinant plasmid pGST-HRV3C-hS100A6 was constructed and transformed into E. coli BL21, then induced by IPTG. After lysed by ultrasound, the fusion protein GST-HRV3C-hS100A6 was purified by Glutathione-Sepharose 4B beads（ GS4BB）, next, digested by GST-HRV3C. the pure recombinant hS100A6 （rhS100A6） was harvestd after removing GST and GST-HRV3C by GS4BB and then identified by Western blot and quantitatied by Spectrophotometry. MTT assay and Hoechst staining were used to detect proliferation and apoptosis of human osteosarcoma cell line 143B, respectively, transwell was used to detect migration and invasion, Western blot was used to detect expression of β-catenin. Results： rhS100A6 was successfully prepared and the protein yield was about 4 mg / L broth. 30p, g/ml of rhS100A6 could promote cell proliferation, migration, invasion and expression of β-catenin （ P 〈 0.05 ） , meanwhile had no significant effect on apoptosis of 143B cell line（ P 〉 0.05 ）. Conclusion ： rhS100A6 is successfully prepared, 30μg/ml of rhS100A6 has promotive effects on human osteosarcoma cell line 143B.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2012年第12期1-7,共7页
China Biotechnology
基金
国家自然科学基金面上项目(30772548)资助项目
关键词
hS100A6
蛋
白
制备
骨肉瘤
Recombinant hS100A6 protein Preparation Osteosarcoma
