冷冻电子显微镜观察人釉原蛋白的自组装  

Observation of human amelogenin self-assembly by cryogenic transmission electron microscopy

作　　者：赖婷婷 陈亮 张川 田鲲[3] LAI Tingting;CHEN Liang;ZHANG Chuan;TIAN Kun(School of Stomatology,Southwest Medical University,Luzhou 646000,China;School of Stomatology,Zunyi Medical University;Sichuan Provincial People's Hospital,Department of Prosthodontics)

机构地区：[1]西南医科大学口腔医学院,泸州646000 [2]遵义医科大学口腔医学院 [3]四川省人民医院口腔修复科

出　　处：《实用口腔医学杂志》2021年第1期114-118,共5页Journal of Practical Stomatology

基　　金：四川省科技厅应用基础项目(编号:2018JY0057)。

摘　　要：目的:采用冷冻电子显微镜(cryo TEM)体外观察人釉原蛋白的自组装过程,分析自组装各阶段蛋白的聚集状态及细微结构。方法:提取青少年下颌第三磨牙牙胚细胞总RNA,用RT-PCR获得人釉原蛋白基因全长,再与pMD19-T载体构建重组质粒,转入宿主菌E.coli Top10中诱导表达并纯化。冷冻电子显微镜观察pH值从3.5跃升至8.0时釉原蛋白发生自组装的聚合状态。结果:当pH值为8.0时在1~20 min内观察到釉原蛋白由低聚体逐步组装成多聚体、纳米球及纳米链等结构。结论:在适宜条件下,人釉原蛋白能分级组装成纳米球。Objective:To observe the self-assembly process of human amelogenin by cryogenic transmission electron microscopy(cryo TEM)in vitro.Methods:Total RNA was extracted from the adolescent's tooth germ of mandibular third molar,and the amelogenin cDNA was obtained from the total RNA through full-length gene of amelogenin by RT-PCR.A plasmid was constructed and inserted into pMD19-T vector,then it transformed into E.coli Top10 to acquire purified proteins.cryo TEM was used to observe and record the self-assembling of amelogenin at pH 3.5 to pH 8.0.Results:When pH=8.0,amelogenin gradually assembled from oligomers into multimers,nanospheres and nanochains within 1-20 min.Conclusion:Under suitable conditions,human amelogenin may self-assemble into nanospheres in stages.

关 键 词：冷冻电子显微镜 釉原蛋白 自组装 

分 类 号：R780.2[医药卫生—口腔医学]