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机构地区:[1]上海交通大学医学院附属新华医院、上海市儿科医学研究所,200092
出 处:《中华医学遗传学杂志》2013年第6期673-677,共5页Chinese Journal of Medical Genetics
基 金:上海市政府(2008ZD001、08JC1416100、09QA1404700、2007145);国家高技术发展计划(2007AA022447);国家科技支撑计划(2006BA105A09)
摘 要:目的对苯丙氨酸羟化酶(phenylalaninehydroxylase,PAH)基因7种突变(R270G、P275A、F121L、A156P、E183G、1324N和R408Q)进行功能分析,通过检测其蛋白表达及酶活性的变化,探讨突变效应和致病性质,进一步明确基因型和表型之间的关系。方法(1)应用体外定点诱变技术构建含有7种突变型PAHcDNA的表达载体,转化感受态大肠杆菌,提取质粒并测序验证。(2)将含有野生型和突变型PAHcDNA的真核表达载体pcDNA3.0瞬时转染COS-7细胞,转染48h后提取总蛋白,应用免疫印迹法检测蛋白表达量并进行酶活性分析。以野生型PAH作为参照,计算突变型PAH的蛋白表达量、残余酶活性。结果R270G、P275A、F121L、A156P、E183G、1324N和R408Q突变型PAH的蛋白表达量分别为野生型的10.5%,56.6%,54.3%,8.70o,8.5%,67.3%和85.4%。体外表达酶活性分别为野生型的7.7%,27.6%,19.0%,10.4%,9.1%,50.6%和40.2%。结论7种突变型PAH的体外表达量及酶活性相对于野生型均有明显降低,证实这7种突变均为引起PAH酶活性降低的致病性突变。Objective To study the in vitro expression of 6 novel missense mutations (R270G, P275A, F121L, A156P, E183G, I324N and a previously described R408Q mutation of phenyialanine hydroxylase (PAH) gene and explore the genotype-phenotype correlation through comparison of protein levels and residual enzyme activities. Methods Seven expression vectors containing PAH cDNA were constructed with a site-directed mutagenesis kit. The plasmids were extracted and sequenced to confirm the target mutations, pcDNA3. 0 containing PAH cDNA was transfected into COS-7 cells and total proteins were extracted 48 h after transfection. The quantities o{ proteins and residual enzyme activities of the 7 mutants were assessed with the wild-type PAH gene as reference. Results Relative quantities of PAH proteins for R270G, P275A, F121L, A156P, E183G, I324N and R408Q were 10.5%, 56.6%, 54.3%, 8.7%, 8.5%, 67. 3% and 85. 4%, respectively. The residual enzyme activities were 7. 7%, 27. 6%, 19.0 %, 10.4 %, 9.1%, 50.6 % and 40.2 %, respectively. Conclusion PAH residual enzyme activities of 7 PAH mutants were all significantly reduced.
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