Isolation and functional analysis of genes uniquely expressed in human corneal epithelium and conjunctival epithelium

我们构建了人角膜上皮mRNA表达谱，并分析了角膜上皮中大量表达或特异表达的基因。从这些数据中，一种新的组织蛋白酶（组织蛋白酶V），尿斑蛋白Ib和Cl通道被分离和分析，其功能和染色体定位。组织蛋白酶V（Cathepsin V）是一种新的组织蛋白酶，其ORF与人组织蛋白酶L（Cathepsin L）的同源性为77%。使用杆状病毒表达系统产生的重组组织蛋白酶V蛋白具有半胱氨酸蛋白酶的蛋白水解活性。RT-PCR结果显示，仅在角膜中，组织蛋白酶V的表达水平高于组织蛋白酶L。组织蛋白酶V可能在角膜生理中起重要作用。用FISH技术将组织蛋白酶V基因定位于9q22.2，距组织蛋白酶L基因15 cM处。这表明，组织蛋白酶L和V的进化更快， 关于我们 通过基因复制从祖先基因中分离出来。Uroplakin Ib蛋白具有4个跨膜结构域。该克隆是已经发表的尿斑蛋白Ib的同种型，因为该克隆的3 '-UTR与已发表的尿斑蛋白Ib不同。RT-PCR结果显示，尿斑蛋白Ib mRNA不仅在移行上皮中表达，而且在眼表也有表达。尿斑蛋白Ib蛋白免疫组化结果显示，尿斑蛋白Ib蛋白分布于角膜、角膜缘和结膜上皮细胞膜，尤其是角膜上皮层的表浅部。一个新的C1通道编码943个氨基酸，定位于染色体1 p32区域。C1通道mRNA的丰度是其他C1通道的100倍，是角膜透明性的一个重要规律，正常结膜上皮的表达谱与角膜上皮有很大的不同。在该表达谱中，角蛋白13、β-2微球蛋白和脂皮质素高度表达。在三个或更多克隆中出现的丰富转录本中，包括两个未知的结膜特异性基因。少

We constructed the mRNA expression profile from human corneal epithelium and analyzed the genes expressed abundantly or specifically in corneal epithelium. From these data, a novel cathepsin (cathepsin V), uroplakin Ib and Cl channel were isolated and analyzed as to their functions and chromosomal localizations. The expression profile of human conjunctival epithelium was also constructed and compared to that of human corneal epithelium.Cathepsin V was a novel cathepsin, which contained ORF of 77% identical homology to human cathepsin L. A recombinant cathepsin V protein produced using a baculovirus expression system has proteolytic activity as a cysteine proteinase. By RT-PCR, only in cornea, the expression level of cathepsin V was higher than that of cathepsin L. Cathepsin V may play an important role in corneal physiology. By the FISH method, cathepsin V genes was mapped to chromosomal region 9q22.2, 15cM from the cathepsin L gene. This suggests that cathepsin L and V evolved more re … More cently by gene duplication from an ancestral gene. Uroplakin Ib protein had four transmembrane domains. This clone was an isoform of uroplakin Ib, which had already published, because a 3'-UTR of this clone was differed from published uroplakin Ib. By RT-PCR, uroplakin Ib mRNA was detected not only in transitional epithelium, but also on the ocular surface. By immunohistochemistry using antiserum against uroplakin Ib peptide, uroplakin Ib protein was found in cell membranes of corneal, limbal and conjunctival epithelium, especially in the superficial half of the corneal epithelial layer. A novel C1 channel coded 943 amino acids and mapped to chromosomal region 1p32. C1 channel mRNA was 100 times more plentiful than other C1 channels, implying an important rule of corneal transparency.The expression profile of normal conjunctival epithelium was so differed greatly from those of corneal epithelium. In this expression profile, keratin 13, beta- 2 microglobulin and lipocortin were highly expressed. Among the abundant transcripts appearing in three or more clones, two unknown conjunctival specific genes were included. Less