- 作者: 李桂楨; 王政光; 黃淑芬; 戴坤蓉
- 作者服務機構: 臺灣師範大學生物學系
- 中文摘要: 本論文在探討中國人族群的多型性DNA單套型與IDUA酵素活性的相關性。我們自正常人族群逢機取樣85人,萃取其基因組DNA,放大包含A8、Q33H(表現子1)、R105Q(表現子3)、A361T(表現子8)或V454I(表現子9)等多型性點的DNA片段,再以限制片段長度多型性(RFLP)或對偶基因專一的寡核甘酸引子(ASO),來檢測各樣品的多型性基因型。我們並自上述各血液樣品中分離白血球,測其IDUA酵素活性。結果發現各樣品的IDUA酵素活性差異頗大(約介於50~300 nmol/h/mg cell protein間),其平均值約156 nmol/h/mg cell protein。在酵素活性高於110%平均值的樣品群中,Q33H的不具多型性的對偶基因“1”及R105Q、A361T、V4541的多型性對偶基因“2”的頻率較酵素活性低於90%平均值的樣品群為高。單套型的連鎖不平衡分析顯示,R105Q、A361和V454I的多型性對偶基因間呈現連鎖關係。當檢測Q33H、R105Q、A361T及V454I的單套型時,單套型1,2,2,2 (方程式無法摘錄) 和IDUA酵素活性有相關性。帶有R105Q、A361T或V4541多型性的IDUA cDNA,於COS-7細胞中所表現的酵素活性,均較正常cDNA高。若將 (方程式無法摘錄) 等和酵素活性正相關的多型性集中於一cDNA上,則或可表現出一具高活性的IDUA蛋白,應用於MPSI患者的酵素替換治療上。
- 英文摘要: The correlation of polymorphic DNA haplotype of the α-L-iduronidase (IDUA) gene and IDUAactivity in Chinese subjects was investigated. Genomic DNA extracted from 85 randomly sampled normalindividuals was used to amplify fragments containing the polymorphic change site A8, Q33H (exon 1), R105Q(exon 3), A361T (exon 8), or V454I (exon 9). The PCR amplified products were analyzed by means ofrestriction fragment length polymorphism (RFLP) or allele specific oligonucleotide (ASO) hybridization.Leukocytes were isolated from the above 85 samples, and their IDUA activities were determined. A widerange of IDUA activity (50~300 nmol/h/mg cell protein) with an average of 156 nmol/h/mg cell protein wasobserved. When the allele frequency was compared between individuals with IDUA activity below 90%or above 110% of the average, a bias toward the common allele “1”of Q33H (方程式無法摘錄) was detected in indi-viduals with higher IDUA activity. Conversely, the polymorphic allele“2”of R105Q (方程式無法摘錄) ,A361T(方程式無法摘錄), and V454I (方程式無法摘錄) was found in the higher IDUA activity group. Linkage disequilibrium analysisof the haplotype data revealed strong nonrandom association among the polymorphic alleles of R105Q,A361T, and V454I. Of the haplotypes constructed by Q33H, R105Q, A361T, and V454I, a positive correlationbetween haplotype 1,2,2,2 (方程式無法摘錄) and IDUA activity was observed. The IDUA activitywas found to increase with (方程式無法摘錄) polymorphic changes by mutagenesis and expression ofIDUA cDNA in COS-7 cells. By combining the positive effect of (方程式無法摘錄) and (方程式無法摘錄) in one cDNAconstruct, it may be possible to produce a high activity IDUA protein for MPS I enzyme replacement therapy.
- 中文關鍵字: IDUA polymorphism; haplotype; α-L-Iduronidase activity.
- 英文關鍵字: --