摘要
目的:获得高纯度的重组人成釉蛋白C端肽。方法:实验于2001-01/2004-06在解放军第四军医大学口腔内科实验室解放军第四军医大学基础部生物化学与分子生物学实验室完成。①转化pRSET-A/成釉蛋白原核表达质粒入BL21(DE3)细菌进行大量培养,加入诱导剂IPTG至终浓度为0.1mmol/L,32℃继续振荡诱导培养5h,收集细菌,诱导菌体的裂解上清经金属螯合亲和层析,洗脱Ni-NTA结合蛋白,收集洗脱蛋白,聚丙烯酰胺凝胶电泳法鉴定。②离子交换层析缓冲液(pH 7.4,50 mmol/L Tris·Cl)平衡Q SapharoseHP层析柱,将初步纯化的蛋白样品加在Q Sapharose HP凝胶介质上,梯度洗脱结合的蛋白,收集洗脱峰,制样,聚丙烯酰胺凝胶电泳法鉴定洗脱蛋白。结果:①表达重组人成釉蛋白C端肽的金属螯合亲和层析结果:诱导菌体的裂解上清经金属螯合亲和层析,洗脱下Ni-NTA结合蛋白,经聚丙烯酰胺凝胶电泳法电泳证实获得初步纯化的重组人成釉蛋白C端肽,其中仍含一些杂蛋白。②重组人成釉蛋白C端肽的离子交换层析结果:获得多个蛋白洗脱峰,聚丙烯酰胺凝胶电泳法证实获得纯化的重组人成釉蛋白C端肽。结论:获得了纯化的人成釉蛋白重组蛋白。
AIM： To obtain high purified Ctelopeptide of recombinant human ameloblastln.
METHODS： The experiment was conducted in the Laboratory of Stomatology and Laboratory of Biochemistry and Molecular Biology, Basic Department of Fourth Military Medical University of Chinese PLA from January 2001 to June 2004, ① The prokaryotic expression plasmid of ameloblastin pRSET-A was put into the BL21 （DE3） germ for large amount of culture, which was induced by IPTG inductor until reached to concentration of 0.1 mmol/L, and the shaking culture was continued at 32 % for 5 hours. The germ was collected, and the split supematant Of inducing germ was treated by metal chelate affinity chromatography, and the Ni-NTA compound protein was eluted, which was then collected to identified by polyacrylamide gel electrophoresis.②The buffer solution of ion exchange chromatography （pH 7.4, 50 mmol/L Tris.C1） was used to balance the chromatographic column of Q Sapharose HP. Primarily purified protein sample was added into Q Sapharose HP gel media, and the compound protein was eluted with gradient, and the eluting peak was collected, and the samples were established. The eluted protein was identified by polyacrylamide gel electrophoresis.
RESULTS： ① The results of metal chelate affinity chromatography of Ctelopeptide of recombinant human ameloblastin： The split supematant of inducing germ was identified to be pnmarily purified C telopeptide of recombinant human ameloblastin by polyacrylamide gel electrophoresls after metal chelate affinity chromatography and elution of Ni- NTA compound protein, while there was still some hybddprotein in it. ③ The ion exchange laminar analysis of C telopeptide of recombinant human ameloblastin： multiple protein eluting peak were obtained and it was proved by polyacrylamide gel electrophoresis that purified C telopeptide of recombinanthuman ameloblastin was obtained.
CONCLUSION： Purified C telopeptide of recombinant human ameloblastin is obtained.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第2期219-221,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助(30300386)
全军"十五"计划基金重点课题(012089)
第四军医大学学术新人资助基金资助~~
