人牙胚釉原蛋白成熟肽基因的克隆  

Cloning of human amelogenin gene encoding mature peptide

作　　者：程岚[1] 雷建强[2] 朱祺泉[2] 王洪海[2] 束蓉[1] 

机构地区：[1]上海第二医科大学附属第九人民医院口腔医学院口腔内科,上海200011 [2]复旦大学,上海200423

出　　处：《上海口腔医学》2004年第2期126-129,共4页Shanghai Journal of Stomatology

基　　金：上海市卫生局科技发展基金

摘　　要：目的:克隆人釉原蛋白成熟肽编码区基因片断,并构建含有该基因的重组表达质粒。方法:从引产胎儿牙胚组织中抽提总RNA,以Oligo(dt)为引物,逆转录合成牙胚cDNA利用PCR,从cDNA中扩增出人釉原蛋白成熟肽编码序列(约540bp),所得目的基因片断插入表达载体质粒PQE30,转化到大肠杆菌DH5α后挑选阳性克隆,抽提重组质粒DNA,通过PCR、酶切和核苷酸序列分析,鉴定阳性克隆。结果:样品质粒测序证实,质粒PQE30中插入的基因片段与人釉原蛋白成熟肽基因序列完全相同。结论:从人胚胎的牙胚组织中克隆到釉原蛋白成熟肽编码序列,成功构建含有人釉原蛋白成熟肽基因的重组表达质粒。PURPOSE:The purpose of this study was to clone human amelogenin gene encoding mature protein, which provides a basis for expressing the recombinant human amelogenin in Escherichia coli. in the future. METHODS: In this study, total RNA was extracted from the dental germ of a legally aborted embryo by Trizol. Using RT-PCR technique we obtained synthesis of cDNA from the total RNA, and the desired DNA products were conducted with PCR from cDNA. The segment was inserted into expression vector PQE30 and the interesting plasmid was transformed into Escherichia coli. host DH5α. The double-stranded DNA of positive clone was analyzed by PCR, restriction endonuclease mapping and DNA sequence analysis. RESULTS: The sequence analysis of recombinant plasmid showed that the human amelogenin encoding mature protein was inserted into vector PQE30 accurately. CONCLUSIONS:We conducted human amelogenin encoding mature protein from dental germ of a legally aborted embryo and got the recombinant plasmid which may express amelogenin gene for further research.

关 键 词：釉原蛋白 逆转录多聚酶链式反应 重组质粒 PCR 牙胚组织 基因克隆 成熟肽基因 

分 类 号：R780.2[医药卫生—口腔医学]