【摘要】 目的构建、表达、纯化、鉴定并标记TAT-Smad7-HA融合蛋白,验证其在体外培养的人原代瘢痕疙瘩成纤维细胞(keloid fibroblast cell,KFB)中的转导活性。方法分别将TAT-PTD、Smad7基因和HA片段依次连接入pET32a(+)质粒构建成pTAT-Smad7-HA重组原核表达载体,IPTG诱导表达后经亲和纯化、Western blot验证、肠激酶切割、目的片段回收和FITC标记后,将FITC-TAT-Smad7-HA融合蛋白作用于体外培养的人原代KFB以观察其转导活性。结果成功构建了TAT-Smad7-HA融合蛋白的原核表达载体pTAT-Smad7-HA,目的蛋白在诱导后获得了高效表达(占菌体总蛋白的25%以上),并成功进行了纯化(纯度达95%以上)、脱盐柱脱盐、Western blot验证、硫氧环蛋白切除及FITC标记,得到相对分子质量约为50×103的FITC-TAT-Smad7-HA融合蛋白,并进一步在体外培养的人KFB细胞中证实了其高转导活性。结论本实验获得了高纯度的FITC-TAT-Smad7-HA融合蛋白,并验证了其在KFB中的高转导活性。更多还原

【Abstract】 Objective To construct,express,purify,identify and label the TAT-Smad7-HA fusion protein (protein transduction domain of trans-activator,human Smad7 and hyaluronic acid) and to validate its transduction activity in the cultured human primary keloid fibroblast cells. Methods TAT-PTD,Smad7 and HA fragments were sequentially inserted into pET32a(+) to construct the pTAT-Smad7-HA prokaryotic expression vector. After the expression of target fusion protein was induced to express by IPTG,affinity purification,Western blot analysis,enterokinase cleavage,target protein capture and FITC labeling were subsequently performed by turns to obtain the FITC-TAT-Smad7-HA fusion protein and to further observe its transduction activity in the human primary KFB cells in vitro. Results The prokaryotic expression vector for the TAT-Smad7-HA fusion protein,named as pTAT-Smad7-HA was successfully constructed,and the target fusion protein was efficiently induced to express,covering more than 25% of the total bacterial proteins,successfully purified with a purity of more than 95% purity,desalted by desalting column,identified by Western blotting,thioredoxin removed and FITC labeled. Finally,a fusion protein of FITC-TAT-Smad7-HA with the approximately molecular weight of 50×103 was successfully purified and its high transduction activity in KFB cells was validated. Conclusion The highly-purified FITC-TAT-Smad7-HA fusion protein and the validation of its high transduction activity in KFB cells have provided an experimental foundation for further studies on the role the human Smad7 protein playing in the TGF-β/Smads signal transduction pathway and further elucidation of the pathogenesis of keloid formation.更多还原