人釉原蛋白基因的扩增及其真核表达克隆的构建

目的 构建人釉原蛋白基因真核表达克隆。方法 从胚龄20周的引产胎儿牙胚中提取总RNA，RT-PCR法扩增人釉原蛋白编码区基因，重组到真核表达载体PcDNA3．1中，经酶切和DNA序列分析验证。结果 经RT-PCR扩增后，得到大小约570bp的特异性产物，与预期的人釉原蛋白mRNA编码区碱基长度一致，重组克隆PcDNA3．1-AMG的测序结果显示，与GenBank中的人釉原蛋白基因(AMELX)序列仅在第485位碱基发生错配(G→C)，但不影响氨基酸组成。结论 本研究成功的构建了人釉原蛋白基因真核表达克隆，为进一步研究牙周组织再生的基因治疗奠定了基础。

Construction of Eukaryotic Expression Clone for Human Amelogenin

Objective To construct the eukaryotic expression clone for human amelogenin. Methods Total RNA was isolated from human fetal tooth buds. RT-PCR was used to amplify the amelogenin encoding region, and the amplified fragment for human amelogenin was inserted into eukaryotic expression vector PcDNA 3.1. The positive clones were selected and analyzed by restriction endonuclease mapping and DNA sequencing. Results 570 bp fragment was produced by RT-PCR; it was of the same size as expected based on human ameloginin mRNA encoding area length. The sequence of the inserted fragment from the recombinant clone PcDNA 3.1-AMG was consistent with that of AMELX from GenBank with one mismatch on 485 from G to C, without affecting the amino acid sequence. Conclusion The eukaryotic expression clone PcDNA3.1-AMG was successfully constructed with the properly inserted DNA sequence encoding mature human amelogenin.