Sawamura D, Li K H, Nomura K, Sugita Y, Christiano A M, Uitto J
Department of Dermatology, Jefferson Medical College, Philadelphia, Pennsylvania.
J Invest Dermatol. 1991 Jun;96(6):908-15. doi: 10.1111/1523-1747.ep12475433.
A human epidermal keratinocyte lambda gt11 recombinant cDNA library was screened with a 0.45-kb cDNA that was generated by polymerase chain reaction (PCR) amplification of a segment of human bullous pemphigoid (BP) antigen mRNA. The screen yielded five clones, the largest one, pcBPA-4, being 2.3 kb in size. The pcBPA-4 cDNA hybridized in Northern analyses with an approximately 9-kb mRNA from cultured keratinocytes, whereas no hybridization signal was detected with RNA from human skin fibroblast, fibrosarcoma HT-1080 cells, or amniotic epithelial WISH cell cultures. Nucleotide sequencing of pcBPA-4 revealed an open reading frame encoding a putative polypeptide of 447 amino acids. This polypeptide showed 88% homology with corresponding mouse BP antigen sequences, and a region of it was identical to a segment in previously published human BP antigen sequences. The 2.3-kb cDNA isolated here was different from a previously published human BP antigen cDNA, in that the open reading frame coded for 264 additional amino acids at the carboxyl end of the putative polypeptide. Known human BP antigen amino acid sequences, compared with mouse sequences, were predicted to be moderately divergent with a unit of evolutionary period (UEP) of 4.5 millions of years (MY). Southern hybridizations suggested that the BP antigen gene (BPAG1) is present as a single copy in the human genome. Southern analyses also revealed the presence of a StuI restriction fragment length polymorphism that can be used for linkage analyses to study the inheritance of a particular BPAG1 allele and a heritable cutaneous disorder affecting the basement membrane zone, such as epidermolysis bullosa.
用人大疱性类天疱疮（BP）抗原mRNA片段通过聚合酶链反应（PCR）扩增产生的0.45kb cDNA筛选人表皮角质形成细胞λgt11重组cDNA文库。筛选得到5个克隆，最大的一个pcBPA - 4大小为2.3kb。pcBPA - 4 cDNA在Northern分析中与培养的角质形成细胞中约9kb的mRNA杂交，而在人皮肤成纤维细胞、纤维肉瘤HT - 1080细胞或羊膜上皮WISH细胞培养物的RNA中未检测到杂交信号。pcBPA - 4的核苷酸测序揭示了一个编码447个氨基酸的推定多肽的开放阅读框。该多肽与相应的小鼠BP抗原序列显示出88%的同源性，并且其一个区域与先前发表的人BP抗原序列中的一个片段相同。此处分离的2.3kb cDNA与先前发表的人BP抗原cDNA不同，在于开放阅读框在推定多肽的羧基末端编码另外264个氨基酸。将已知的人BP抗原氨基酸序列与小鼠序列比较，预测其在进化周期单位（UEP）为450万年（MY）时具有中等程度的差异。Southern杂交表明BP抗原基因（BPAG1）在人类基因组中以单拷贝形式存在。Southern分析还揭示了一种StuI限制性片段长度多态性的存在，可用于连锁分析以研究特定BPAG1等位基因的遗传以及影响基底膜带的遗传性皮肤疾病，如大疱性表皮松解症。
