【作者】 宋璐璐；

【导师】 蒋玲；

【作者基本信息】 山东大学 ， 内科学， 2008， 硕士

【摘要】 第一部分突变CYP21基因表达载体的构建目的:21-羟化酶缺陷症(21-hydroxylase deficiency,21-OHD)是最常见的先天性肾上腺皮质增生(congenital adrenal hyperplasia,CAH),呈常染色体隐性遗传,约占全部病例的90-95%。本课题组在2个中国汉族CAH患者家系的基因分析中检测到两种错义突变,其中一个为首次报道的新突变P459H,另一个为稀有突变R483W,为明确两个点突变的意义以及进行基因型-表现型关联分析,我们采用体外实验的方法进行了功能学研究。首先对野生型CYP21cDNA分别进行P459H和R483W点突变,构建分别携带P459H和R483W突变的真核表达载体。方法:1.以质粒pCMV4-CYP21作为模板,分别设计两组特异引物,用高保真PfuDNA聚合酶催化PCR反应,在野生型CYP21基因上引入突变P459H或R483W,构建分别携带突变CYP21的真核表达载体:pCMV4-CYP21-R483W和pCMV4-CYP21-P459H。2.产物双向测序鉴定。结果:1.对突变产物进行BglⅡ和KpnⅠ双酶切,所得条带大小约为1800bp和4800bp。2.通过DNA双向测序证实,除导入的特异突变位点外无其它新的突变产生,突变的pCMV4-CYP21表达载体构建成功。结论:采用高保真Pfu DNA聚合酶,设计特定引物,可以成功引入目的基因突变,构建突变CYP21的重组表达载体,为体外研究点突变的意义提供了基础。第二部分CYP21基因突变的体外功能学研究目的:通过体外试验对两个错义突变P459H和R483W进行功能学研究,明确它们的的意义以及进行基因型-表现型关联分析。方法:分别将携带突变P459H和R483W的表达载体——pCMV4-CYP21-P459H和pCMV4-CYP21-R483W转染COS-7细胞,待其表达24～48h后,在培养基中加入其天然底物孕酮,检测产物11-DOC的产量,并计算不同CYP21蛋白催化孕酮生成11-DOC的转化率。以WT-GYP21的孕酮转化率作为100%,将突变CYP21的孕酮转化率与其作比,以所得比值来表示其残余酶活性。结果:1.点突变P459H和R483W都显著降低了CYP21对孕酮的催化活性。P459H突变的CYP21在体外实验中部分失活,残存的催化活性为6.8%(SD=2.1%),而R483W突变后的CYP21对孕酮的催化活性丧失至2.9%(SD=1.5%)。所有数据均经过student’t检验,p＜0.05。2.突变蛋白的三维结构分析显示459位脯氨酸位于一个袢环结构,P459H导致脯氨酸被置换为组氨酸后,可能与L螺旋的氨基酸残基L464或Q477发生相互作用,从而在一定程度上影响蛋白功能;R483W突变导致483位的精氨酸变成色氨酸后,可能与J螺旋的322位酸性氨基酸天冬氨酸及氨基酸残基以及L445或L446之间的相互作用消失从而影响蛋白结构。结论:1.P459H和R483W都显著降低CYP21的功能。体外实验结果显示两个点突变残余活性分别为6.8%和2.9%。2.根据基因不便导致的CYP21活性改变分类,P459H和R483均属于单纯男性化组,两个21-OHD病例的基因型和表现型一致。3.P459H和R483W突变改变了蛋白的三维结构或功能,降低了酶活性,这为分析CYP21结构-功能的关系提供了新的视点。更多还原

【Abstract】 1.Site-directed mutagenesis of CYP21Objectives:21-hydroxylase deficiency(21-OHD)is the most frequent cause of congenital adrenal hyperplasia,a group of inherited metabolic disorders.Clinically,21-OHD is classified into three types:salt-wasting,simple-virilizing and non-classic.Deletion or mutations of CYP21(gene encoding 21-hydroxylsae)account for the disease. Generally there is good correlation between phenotype and genotype of 21-OHD,but exceptions also happen.Recently,by direct sequencing of the whole CYP21 gene of two simple-virilizing patients,a novel mutation P459H and a rare mutation R483W were detected.In order to determine how much enzyme activity are damaged respectively by each of the two mutations and then make genotype-phenotype correlation analysis,we did in vitro expression and assays of enzyme activity.First,we must introduce P459H and R483W mutation respectively into wild-type CYP21 and reconstruct plasmids containing mutant CYP21.Methods:To construct mutated CYP21,PCR reactions were done with template wide-type CYP21 and specially designed primers.Pfu DNA polymerase was used to avoid additional mutations.This is site-directed mutagenesis method.Finally the constructs was sequenced to confirm target mutations.Results:(1)Mutated pCMV4-CYP21 was digested by two restriction enzymes BglII and KpnI,which produced two fragments of approximately 1800bp and 4800bp.(2)Sequencing of mutated constructs verified target mutations but no additional mutaions. Conclusions:Plasmids with P459H or R483W mutated CYP21 were constructed by site-directed mutagenesis and can be used to express mutated CYP21 enzyme proteins.2.Functional analysis of mutated CYP21Objectives:To determine enzyme activity of P459H and R483 W mutated CYP21 and make genotype-phenotype correlation analysis.Methods:1.Transient expression of plasmids containing CYP21 in COS-7 cells.2.~3H-labeled progesterone was added to each well as the substrate to test enzyme activities.Results:1.As the result of three independent experiments,the two missense mutations P459H and R483W both show harm to 21-hydroxylase activity,as shown in Fig.2.With the WT CYP21 activity is defined as 100%,the residual activity toward progesterone of P459H mutated protein is reduced to 6.8%(SD=2.1%)of WT 21-hydroxylase,while R483W mutant was reduced to 2.9%(SD=1.5%)(P＜0,05,Student’s t test)2.P459 is highly conservative in all orthologs known today,and is deeply buried in the inner core of the protein.When praline is substituted by histidine,interreactions between P459 and L464 or Q477 are lost,which may influence function of the protein.R483 is also highly conserved.Substitution of arginine by tryptophane destroyes interreactions between R483 and D322 of helix J as well as L445 and L446.Conclusions:The results revealed that both mutanted CYP21 significantly reduced enzyme activity towards progesterone.Thus the phenotype -genotype correlation of the two patients was well established.We also tried to explain their partial 21-hydroxylase activity loss by constructing three-dimensional model of mutated CYP21.The study had evident significance to predicting severity of the disease and to family genetic consultation.更多还原