Guise K S, Korneluk R G, Waye J, Lamhonwah A M, Quan F, Palmer R, Ganschow R E, Sly W S, Gravel R A
Gene. 1985;34(1):105-10. doi: 10.1016/0378-1119(85)90300-2.
Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of beta-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73%. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropyl-thio-beta-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.
VII型黏多糖贮积症是一种由于β-葡萄糖醛酸酶（BG）活性缺乏导致的溶酶体贮积病。为便于对该疾病的突变进行研究并为患病家庭提供分子诊断工具，我们分离了人BG cDNA克隆。用小鼠BG cDNA克隆（pGUS-1）的一个片段筛选了冈山和伯格构建的SV40转化的人成纤维细胞cDNA文库[《分子细胞生物学》3 (1982) 280 - 289]。通过限制性酶切图谱分析，分离得到的17个人cDNA克隆（pHUG）是相同的，只是长度有所不同。在一个198 bp的PvuII - SstI限制性片段中，pHUG克隆与pGUS-1的DNA序列同源性为80%。在测序区域，pGUS-1和pHUG克隆均含有一个开放阅读框（ORF），预测的氨基酸序列同源性为73%。将亚克隆到pUC9中的pHUG-1的1150 bp片段在大肠杆菌中表达，产生了一种异丙基硫代-β-半乳糖苷（IPTG）诱导型的35 kDal融合蛋白，该蛋白可被山羊抗人BG免疫球蛋白G（IgG）特异性免疫沉淀。这一证据直接证实了pHUG cDNA克隆对应于人BG。
