摘要目的 对苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)基因7种突变(R270G、P275A、F121L、A156P、E183G、I324N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PAH cDNA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PAH cDNA的真核表达载体pcDNA3.0瞬时转染COS-7细胞,转染48 h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、I324N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％,8.7％,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.

abstractsObjective To study the in vitro expression of 6 novel missense mutations (R270G,P275A,F121L,A156P,E183G,I324N and a previously described R408Q mutation of phenylalanine 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 of 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.