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

人角膜上皮和结膜上皮独特表达基因的分离和功能分析

基本信息

批准号：
10470365
负责人：
KINOSHITA Shigeru
金额：
$ 8.19万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470365/
关键词：
corneal epithelium conjunctival epithelium human cDNA library specific gene cathepsin V uroplakin Ib Cl channel cDNA解析

项目摘要

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

我们构建了人角膜上皮的 mRNA 表达谱，并分析了角膜上皮中丰富或特异性表达的基因。从这些数据中，分离出一种新型组织蛋白酶（组织蛋白酶 V）、尿斑蛋白 Ib 和 Cl 通道，并分析了它们的功能和染色体定位。还构建了人结膜上皮的表达谱，并与人角膜上皮的表达谱进行比较。组织蛋白酶 V 是一种新型组织蛋白酶，其 ORF 与人组织蛋白酶 L 具有 77% 的同源性。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可能在角膜生理中发挥重要作用。通过FISH方法，将组织蛋白酶V基因定位到距组织蛋白酶L基因15cM的染色体区域9q22.2。这表明组织蛋白酶 L 和 V 是通过祖先基因的基因复制而进化得更加精确。 Uroplakin Ib 蛋白具有四个跨膜结构域。该克隆是已发表的尿斑蛋白 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。通过RT-PCR，不仅在移行上皮中检测到了尿斑蛋白Ib mRNA，还在眼表面上检测到了尿斑蛋白Ib mRNA。通过使用抗尿斑蛋白Ib肽的抗血清进行免疫组织化学分析，在角膜、角膜缘和结膜上皮的细胞膜中发现尿斑蛋白Ib蛋白，尤其是在角膜上皮层的浅半部。一个新的 C1 通道编码 943 个氨基酸并定位到染色体区域 1p32。 C1通道mRNA比其他C1通道丰富100倍，暗示了角膜透明度的重要规则。正常结膜上皮的表达谱与角膜上皮的表达谱有很大差异。在此表达谱中，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。较少的