Characterization of 5 prime flanking region of 230 kDa bullous pemphigoid antigen gene

大疱性类天疱疮抗原(BPA)是大疱性类天疱疮患者血清中循环自身抗体识别的蛋白质。到目前为止，至少有两种抗原被报道，其中一种是230 kDa的蛋白，另一种是180 kDa的胶原蛋白。本研究拟对230 kDa BPA基因的5个碱基侧翼区进行特征分析，以了解该基因的表达调控。首先，我们筛选了含有人230 kDa BPA基因的人和小鼠基因组文库，得到人和小鼠的阳性克隆。对这些克隆的核苷酸序列分析表明，有些克隆含有5个主要侧翼区。长约3kb的5个碱基侧翼区序列检测到了几个可能的顺式作用元件。人和小鼠5个主要侧翼区域的高度序列同源性表明，该基因表达的调节对角质形成细胞的功能至关重要。此外，在从翻译起始点上游扩增人2kb片段和小鼠1.2kb片段后，我们用这些片段构建了氯霉素乙酰转移酶(CAT)，并将它们转染到包括角质形成细胞在内的培养细胞中。仅在角质形成细胞中检测CAT活性表明，这些片段包含负责角质形成细胞特异性表达的顺式元件。我们构建了缺失突变体，并将它们导入角质形成细胞，以检测这些元件的定位。CAT活性表明，角质形成细胞特异性表达的元件位于人5素侧翼区的-216到-197，以及小鼠区域的-525到-213。我们利用该克隆显示了小鼠230 kDa BPA基因的染色体定位，并检测了基质分解素基因启动子中的AP-1位点在紫外线诱导的皮肤变化中的作用，以阐明230 kDa BPA基因5个碱基侧翼区的AP-1位点的功能。

Bullous pemphigoid antigens (BPA) are proteins which are recognized by circulating autoantibodies in the sera of the patients with bullous pemphigoid. At least two antigens have been reported so far ; one of which is a 230 kDa protein, wereas another protein is 180 kDa collagen. This study intended to characterize the 5 prime flanking region of 230 kDa BPA gene in order to know regulation of this gene expression. First, we screened a human and a mouse genomic libraries with a human 230 kDa BPA cDNA,resulting in human and mouse positive clones. Nucleotide sequencing of these clones showed that some clones contained the 5 prime flanking regions. And the sequences of about 3 kb of 5 prime flanking regions detected several putative cis-acting elements. High sequence homology between the human and mouse 5 prime flanking regions suggested that regulation of this gene expression is critical for the function of keratinocytes. Furthermore, after PCR amplification of a human 2kb segment and of a mouse 1.2kb segment upstream from translation starting sites, we made chlolamphenicol acetyltranferase (CAT) constructs with these fragments, and transfected them to cultured cells including keratinocytes. Detection of CAT activity in only keratinocytes indicated that these fragments contained cis-elements responsible for keratinocyte specific expression. We constructed deletion mutants and transfected them to keratinocytes to examine localization of the elements. The CAT activities revieled that the elements for keratinocyte specific expression resided in -216 to -197 of the human 5 prime flanking region, and in -525 to -213 of the mouse region.We made a few studies accompanied with the study mentioned above. We showed chromosomal localization of mouse 230 kDa BPA gene using the mouse clone, and examined a role of AP-1 site in the promoter of stromelysin gene in UV-induced skin changes to clarify the function of the AP-1 site in 5 prime flanking region of 230 kDa BPA gene.