Cloning of Mouse Enamel Matrix Serine Proteinase Encoding Mature Protein

摘要

Objective: To clone cDNA of enamel matrix serine proteinase (EMSP1) encoding mature protein from mouse dental germs. Methods: Total RNA was isolated from developing incisors and molars of 7 days mouse pups and reverse-transcribed into cDNA. Two pairs of specific primers was designed to obtain the desired gene by Touchdown PCR and Nested PCR. The segment was inserted into Vector pMD-18T, and recombined vectors was transformed into E.coli JM109.The positive clone was chose and analysed by restriction endonuclease mapping and DNA sequencing. Results:700 bp of cDNA of mouse EMSP1 was sueccessfully cloned from mouse tooth germs tissue. The sequence was consistent with that displayed in PubMed. Conclusion:The mouse EMSP1 cDNA encoding mature protein is obtained for further study.%目的:克隆小鼠牙胚组织中釉基质丝氨酸蛋白酶(EMSP1)成熟肽编码区基因.方法:提取出生后7 d昆明种小白鼠切牙、磨牙牙胚总RNA,逆转录为cDNA,设计两对特异性引物,采用Touchdown PCR 和嵌套PCR方法,扩增出小鼠EMSP1起始密码子至终止密码子基因片段.将目的基因连入载体pMD-18T,转化入大肠杆菌JM109,通过蓝白筛选,挑选阳性克隆培养扩增,纯化重组质粒进行限制性酶切和核苷酸序列分析鉴定.结果:限制性酶切图谱和核苷酸序列分析均表明所克隆cDNA为小鼠700 bp的EMSP1成熟肽基因编码.结论:成功地克隆了小鼠编码EMSP1成熟肽基因片段.