Lau E C, Bessem C C, Slavkin H C, Zeichner-David M, Snead M L
Calcif Tissue Int. 1987 Apr;40(4):231-7. doi: 10.1007/BF02556627.
To experimentally examine the participation of amelogenins in controlled mineral-phase maturation of mammalian enamel, the identification of the individual proteins and their corresponding gene(s) is required. For this purpose, cDNAs were constructed from polyadenylated RNA from 2-day postnatal murine teeth, molecularly cloned into lambda-gt11 expression vectors and transfected into E. coli. The cDNA library was screened for amelogenin gene(s) by using either antibody or nucleic acid probes. An amelogenin cDNA clone encoding 79 carboxy-terminal amino acid residues and 100 nucleotides of the 3' noncoding sequence was demonstrated to contain a major antigenic site for amelogenin protein by immunostaining of specific amelogenin proteins from total extracted enamel protein blots using clonal epitope selected antibody. This is the first report linking amelogenin epitope(s) to a defined DNA sequence, and consequently a defined portion of the amino acid sequence for amelogenins. Secondary structure analysis, based on the relative average linear hydropathy of the amino acid sequence of amelogenin, predicted epitopes in the amino terminus of the molecule rather than the carboxy terminus. Our present data suggest that the carboxy terminus of the amelogenins is sufficiently externalized to be an antigenic domain. These data may be useful in subsequent structural analysis of amelogenin proteins and enhancing our understanding of their physicochemical participation in biomineralization.
为了通过实验研究釉原蛋白在哺乳动物牙釉质可控矿化相成熟过程中的作用，需要鉴定各个蛋白质及其相应的基因。为此，从出生后2天的小鼠牙齿的聚腺苷酸化RNA构建cDNA，分子克隆到λ-gt11表达载体中并转染到大肠杆菌中。通过使用抗体或核酸探针筛选cDNA文库以寻找釉原蛋白基因。通过使用从总提取的牙釉质蛋白印迹中选择克隆表位的抗体对特定釉原蛋白进行免疫染色，证明一个编码79个羧基末端氨基酸残基和3'非编码序列100个核苷酸的釉原蛋白cDNA克隆含有釉原蛋白的主要抗原位点。这是将釉原蛋白表位与确定的DNA序列以及因此与釉原蛋白氨基酸序列的确定部分联系起来报道。基于釉原蛋白氨基酸序列的相对平均线性亲水性的二级结构分析预测分子氨基末端而非羧基末端存在表位。我们目前的数据表明，釉原蛋白的羧基末端充分暴露在外，使其成为一个抗原结构域。这些数据可能有助于随后对釉原蛋白的结构分析，并增进我们对其在生物矿化中物理化学作用的理解。
