中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2013年
6期
673-677
,共5页
苯丙酮尿症%苯丙氨酸羟化酶%基因突变%体外表达%残余酶活性
苯丙酮尿癥%苯丙氨痠羥化酶%基因突變%體外錶達%殘餘酶活性
분병동뇨증%분병안산간화매%기인돌변%체외표체%잔여매활성
Phenylketonuria%Phenylalanine hydroxylase%Gene mutation%In vitro expression%Residual enzyme activity
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.
目的 對苯丙氨痠羥化酶(phenylalanine hydroxylase,PAH)基因7種突變(R270G、P275A、F121L、A156P、E183G、I324N和R408Q)進行功能分析,通過檢測其蛋白錶達及酶活性的變化,探討突變效應和緻病性質,進一步明確基因型和錶型之間的關繫.方法 (1)應用體外定點誘變技術構建含有7種突變型PAH cDNA的錶達載體,轉化感受態大腸桿菌,提取質粒併測序驗證.(2)將含有野生型和突變型PAH cDNA的真覈錶達載體pcDNA3.0瞬時轉染COS-7細胞,轉染48 h後提取總蛋白,應用免疫印跡法檢測蛋白錶達量併進行酶活性分析.以野生型PAH作為參照,計算突變型PAH的蛋白錶達量、殘餘酶活性.結果 R270G、P275A、F121L、A156P、E183G、I324N和R408Q突變型PAH的蛋白錶達量分彆為野生型的10.5％,56.6％,54.3％,8.7％,8.5％,67.3％和85.4％.體外錶達酶活性分彆為野生型的7.7％,27.6％,19.0％,10.4％,9.1％,50.6％和40.2％.結論 7種突變型PAH的體外錶達量及酶活性相對于野生型均有明顯降低,證實這7種突變均為引起PAH酶活性降低的緻病性突變.
목적 대분병안산간화매(phenylalanine hydroxylase,PAH)기인7충돌변(R270G、P275A、F121L、A156P、E183G、I324N화R408Q)진행공능분석,통과검측기단백표체급매활성적변화,탐토돌변효응화치병성질,진일보명학기인형화표형지간적관계.방법 (1)응용체외정점유변기술구건함유7충돌변형PAH cDNA적표체재체,전화감수태대장간균,제취질립병측서험증.(2)장함유야생형화돌변형PAH cDNA적진핵표체재체pcDNA3.0순시전염COS-7세포,전염48 h후제취총단백,응용면역인적법검측단백표체량병진행매활성분석.이야생형PAH작위삼조,계산돌변형PAH적단백표체량、잔여매활성.결과 R270G、P275A、F121L、A156P、E183G、I324N화R408Q돌변형PAH적단백표체량분별위야생형적10.5％,56.6％,54.3％,8.7％,8.5％,67.3％화85.4％.체외표체매활성분별위야생형적7.7％,27.6％,19.0％,10.4％,9.1％,50.6％화40.2％.결론 7충돌변형PAH적체외표체량급매활성상대우야생형균유명현강저,증실저7충돌변균위인기PAH매활성강저적치병성돌변.
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 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.
