两例RhD--缺失型个体RH基因结构和表达分析
发布时间：2018-08-04 20:44
【摘要】： 目的：RhD--缺失型是Rh血型系统一种极为罕见的变异体,血清学特征表现为红细胞上RhC／c、E／e抗原的完全缺乏和(或)D抗原的过量表达。目前RhD--缺失型的分子生物学机制尚不明确,国外的研究发现了两种情况：一种是RHCE基因完整但无表达活性,另一种是RHCE基因部分缺失。前者考虑是由于mRNA的转录活性降低所致,后者考虑是外显子部分缺失和基因重组两种机制。国内还没有相关报道。由于同一种血型表型,可以对应多种基因型,而且在不同种族和同一种族的不同群体中,同一种表型所对应的基因型也不尽相同。Rh血型系统的多态性一直是阻碍基因定型技术普及的障碍。目前有关血型基因结构的资料,大多数来源于白人,不符合我国的种族群体特点。中国人群民族多,数量大,遗传杂合性大。本研究通过对两例确证为RhD--个体RH基因结构和表达的深入研究,明确中国部分汉族人群的RhD--缺失型个体可能存在的新的发生机制。这对搞清中华民族的血型基因结构,广泛开展血型基因分型技术是十分必要的,还可能发现新的RH等位基因。 方法：(1)从正常人外周血中网织红细胞中提取总RNA逆转录RHD基因；(2)构建RHD基因克隆载体,转染大肠杆菌；(3)地高辛标记PCR法扩增RHcDNA特异性三段探针和Exon1特异性探针,对两例RhD--个体采用HindⅢ和SphⅠ限制性内切酶酶切血液基因组DNA并与特异性探针进行Southern blot,并采用PCR-SSP方法扩增两例RhD--个体RHCE基因多个外显子和内含子；(4)采用RT-PCR方法检测RhD--个体是否有RHCE剪接体转录。 结果：(1)获得RHD基因；(2)构建RHD基因克隆成功；(3)Southern blot发现两例RhD一个体RHCE基因均存在exonl,但存在中间段缺失(exon4-exon7缺失),进一步采用PCR-SSP方法特异性扩增RHCE基因外显子及内含子,证实RhD--个体RHCE intro、exon4-7基因片段丢失,但exon3、exon9及3’端基因序列均存在。(4)未检测到两RhD--个体RHCE基因转录产物表达。 结论：(1)中国部分汉族人群的RhD--缺失型个体的产生原因是RHCE基因exon4-7缺失,其截短的RHCE基因不表达相关活性产物,这与国外学者对白人、黑人RhD--缺失型个体发生的分子机制研究结果不同。
[Abstract]:Objective: the Rh D- deletion is an extremely rare variant of the Rh blood group system. The serological features are the complete absence of RhC / cr / e antigen and / or the over-expression of D antigen on the erythrocytes. At present, the molecular biological mechanism of RhD- deletion type is still unclear. Two situations have been found in foreign studies: one is complete RHCE gene but no expression activity, the other is partial deletion of RHCE gene. The former is due to the decrease of transcriptional activity of mRNA, while the latter is due to two mechanisms: partial deletion of exon and gene recombination. There are no reports at home. Because of the same blood type phenotype, it can correspond to multiple genotypes, and in different groups of different races and the same race, The polymorphism of the Rh blood group system is an obstacle to the popularization of gene typing technology. Most of the information on genetic structure of blood group is white, which does not accord with the ethnic group characteristics of our country. There are many ethnic groups, large numbers and large genetic heterozygosity in Chinese population. In this study, we studied the structure and expression of RH gene in two RhD- individuals confirmed as RhD- individuals, and clarified the possible new mechanism of RhD- deletion type in some Han population in China. It is necessary to understand the blood group gene structure of the Chinese nation and widely develop the blood group genotyping technique, and a new RH allele may also be discovered. Methods: (1) Total RNA reverse transcriptional RHD gene was extracted from reticulocyte of normal human peripheral blood; (2) RHD gene clone vector was constructed and transfected into Escherichia coli; (3) digoxin labeled PCR was used to amplify RHcDNA specific three-segment probes and Exon1 specific probes. Two RhD- individuals were digested with Hind 鈪，
本文编号：2165079
[Abstract]:Objective: the Rh D- deletion is an extremely rare variant of the Rh blood group system. The serological features are the complete absence of RhC / cr / e antigen and / or the over-expression of D antigen on the erythrocytes. At present, the molecular biological mechanism of RhD- deletion type is still unclear. Two situations have been found in foreign studies: one is complete RHCE gene but no expression activity, the other is partial deletion of RHCE gene. The former is due to the decrease of transcriptional activity of mRNA, while the latter is due to two mechanisms: partial deletion of exon and gene recombination. There are no reports at home. Because of the same blood type phenotype, it can correspond to multiple genotypes, and in different groups of different races and the same race, The polymorphism of the Rh blood group system is an obstacle to the popularization of gene typing technology. Most of the information on genetic structure of blood group is white, which does not accord with the ethnic group characteristics of our country. There are many ethnic groups, large numbers and large genetic heterozygosity in Chinese population. In this study, we studied the structure and expression of RH gene in two RhD- individuals confirmed as RhD- individuals, and clarified the possible new mechanism of RhD- deletion type in some Han population in China. It is necessary to understand the blood group gene structure of the Chinese nation and widely develop the blood group genotyping technique, and a new RH allele may also be discovered. Methods: (1) Total RNA reverse transcriptional RHD gene was extracted from reticulocyte of normal human peripheral blood; (2) RHD gene clone vector was constructed and transfected into Escherichia coli; (3) digoxin labeled PCR was used to amplify RHcDNA specific three-segment probes and Exon1 specific probes. Two RhD- individuals were digested with Hind 鈪，
本文编号：2165079
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