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

大疱性类天疱疮抗原（BPA）是一种在大疱性类天疱疮患者血清中被循环自身抗体识别的蛋白质。到目前为止，已经报道了至少两种抗原；一种是230 kDa的蛋白质，另一种是180 kDa的胶原蛋白。本研究旨在对230 kDa双酚a基因的5′侧区进行表征，以了解该基因的表达调控。首先，我们筛选了含有人类230 kDa BPA cDNA的人类和小鼠基因组文库，获得了人类和小鼠阳性克隆。这些克隆的核苷酸测序结果表明，一些克隆含有5 '侧链区域。在5 '侧区约3kb的序列中检测到几个假定的顺式作用元件。人和小鼠5 prime侧区之间的高序列同源性表明，该基因表达的调控对角质形成细胞的功能至关重要。此外，在翻译起始位点上游的人类2kb片段和小鼠1.2kb片段进行PCR扩增后，我们用这些片段构建了氯霉素乙酰转移酶（CAT）构建物，并将其转染到培养的细胞中，包括角质形成细胞。仅在角质形成细胞中检测CAT活性表明这些片段含有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其转染到角质形成细胞中以检测元件的定位。CAT活性表明，角质形成细胞特异性表达的元件位于人类5 '侧区-216至-197，以及小鼠区域-525至-213。在上述研究的同时，我们还做了一些研究。我们利用小鼠克隆对小鼠230 kDa BPA基因进行染色体定位，并检测了stromelysin基因启动子AP-1位点在紫外线诱导皮肤变化中的作用，以阐明230 kDa BPA基因5 prime侧翼区域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.