摘要
目的提取人釉原蛋白全长基因并进行克隆。方法从处于釉质发育阶段的青少年下颌第三磨牙的牙胚中取得细胞总RNA,通过反转录酶-聚合酶链锁反应(reverse transcription-polymerase chain reaction)得到釉原蛋白全长基因,与p MD19-T载体构建质粒后转入大肠杆菌E.coli Top10中扩增克隆。结果经过超微量分光光度计检测,A260/A280为1.99,A260/A230为2.08,表明提取的总RNA质量较高。对釉原蛋白基因与p MD19-T载体构建的质粒进行测序后比对分析,结果与美国国家生物技术信息中心公布的序列一致,表明获得了釉原蛋白全长基因。扩增釉原蛋白全长基因并进行1%琼脂糖凝胶电泳,可见大小约为570 bp的特异性条带,与预期一致。结论可以从处于釉质发育阶段的人第三磨牙牙胚中提取人釉原蛋白全长基因,并能通过与p MD19-T载体质粒构建转入E.coli Top10中成功克隆。
Objective To extract the full-length gene of human amelogenin and then to clone it.Methods Total RNA was extracted from the tooth germs of mandibular third molar,which was in enamel developmental stage.The amelogenin cDNA was obtained from the total RNA through reverse transcription-polymerase chain reaction(RT-PCR).The segments were inserted into pMD19-T vector and the resulted plasmids were transformed into E.coli Top10.The positive clones were sequenced for identification.Results The values of A260/A280 and A260/A230 were 1.99 and 2.08,respectively,through the test of micro-spectrophotometer.The result indicated that we have got the total RNA with high quality.The sequence of plasmids of pMD19-T-Am showed that nucleotide sequence of amelogenin we cloned were consistent with the record of National Center of Biotechnology Information.Amplification of full-length amelogenin gene and 1%agarose gel electrophoresis showed a 570 bp specific band in line with expectations.Conclusion Homo amelogenin has been cloned from total RNA of the tooth germs extracted from mandibular third molars,which is in enamel developmental stage.Human gene of amelogenin is successfully cloned into E.coli Top10 with plasmid pMD19-T vector.
作者
唐渝
姚静
冯小云
田鲲
TANG Yu;YAO Jing;FENG Xiao-yun;TIAN Kun(School of Stomatology,Southwest Medical University,Luzhou 646000,China;Department of Stomatology,Sichuan Provincial People's Hospital,Chengdu 610072,China)
出处
《实用医院临床杂志》
2018年第1期1-4,共4页
Practical Journal of Clinical Medicine
基金
四川省人力资源和社会保障厅留学人员科技活动项目择优资助项目(编号:81100786)
