Kell糖蛋白基因结构与多态性研究
发布时间：2018-10-30 08:44
【摘要】： Kell血型系统是继ABO,Rh血型系统之后最为重要的血型系统。Kell血型系统具有复杂的多态性,到目前为止,共发现了27种抗原,这27种抗原皆由点突变造成。如果所有Kell抗原都不表达,这种特殊的表型称为K-null(K_0)。与野生型k相对应的K抗原是低频率抗原。白种人中,K的抗原频率约为7%。由于K抗原具有较强的免疫原性,所以欧美国家都将Kell作为输血前血型检测的必检项目。中国人群中,Kell血型一直被认为多态性较单一,基本都为k,且K的频率约为0.07%,因此输血前不进行K抗原的定型。至今,国内也很少有Kell血型的相关研究。 我们对中国人群中,K,k,K_0表型的个体进行KEL基因分型研究。分段扩增KEL基因19个外显子,结合测序和克隆测序的方法,分析上述3种表型的基因结构。结果发现,检测的所有K个体的KEL基因均为第6外显子C698T的突变,从而导致Met193Thr氨基酸的改变。k个体的KEL基因与野生型基因序列一致。筛选了87,665例上海地区O型献血员,发现2例K_0个体。这2例K_0个体的KEL基因具有相同的突变类型。一条染色体上第3外显子nt185位具有一个T插入,使得原本的TCT变成TTC,导致Ser62Phe氨基酸改变。并且,因为插入T使阅读框移位,终止密码子在第4外显子提前出现;另外一条染色体,第7外显子nt715位具有G→T突变,使GAA变成TAA,氨基酸发生Glu239Stop的变化。 我们进一步对KEL基因的转录本结构进行了研究。发现在网织红细胞中,k表型以及K表型的个体都具有一条完整的KEL基因转录本,K表型个体可检测到具有突变类型的转录本。但是K_0个体缺乏完整的KEL基因转录本,RNA剪接时采用选择性剪接的模式,包括部分内含子的保留和某些外显子的跳跃。白系细胞中,所有个体的转录本都不止一种,由不同的外显子或内含子通过选择性剪接拼接而成,与K_0个体网织红细胞KEL基因转录本情况类似。 运用流式细胞技术,分析红细胞和白细胞上Kell抗原的表达情况,并区分白系细胞种类,以了解Kell抗原在T淋巴细胞,B淋巴细胞以及NK细胞上的表达情况。流式检测得到结果表明,具有K+或k+表型个体红细胞上均具有很强的Kell抗原表达;而K_0个体的红细胞上没有任何Kell抗原的表达。在白系细胞上,K_0个体同样不表达任何Kell蛋白。但4例k个体各自有不同程度微弱的Kell抗原表达;用4种不同抗体都证实了这一点。进一步的研究发现,具有Kell抗原表达的白系细胞主要为CD4+的T细胞、CD19+的B细胞,而CD8+T细胞、CD56+NK细胞基本没有Kell抗原表达。 对K_0家系的研究证明,先证者的父母Kell表型都正常,各自具有一个突变的KEL基因。造成先证者K_0表型的两条染色体突变基因分别来自其父母。先证者的姐姐遗传得到其母亲的突变基因,另外一条染色体上带有野生型的KEL基因,来自其父亲。因此,她具有正常的Kell表型。先证者的弟弟遗传得到父母正常的染色体,有正常的Kell表型。由于两种不同突变的KEL基因同时遗传给先证者,造成了罕见的K_0表型。而仅仅一条染色体上的KEL基因突变并不能造成Kell表型异常。 K/k分型试剂盒的研制及应用。设计特异性引物,建立序列特异性引物PCR(sequence-specific primer polymerase chain reaction,PCR-SSP)的方法,用于鉴定K及k基因型,并结合测序,鉴定试剂盒的可靠性和重复性。该K/k PCR-SSP基因分型试剂盒成功应用于外籍孕妇产前检查的K/k基因型筛选和2例ISBT(International Society Blood Transfusion)血型基因定型workshop标本的分析。
[Abstract]:The Kell blood type system is the most important blood type system following ABO and Rh blood type systems. The Kell blood group system has a complex polymorphism. So far, 27 antigens have been found, all of which are caused by point mutation. If all Kell antigens are not expressed, this special phenotype is referred to as K-null (K _ 0). The K antigen corresponding to the wild type k is a low frequency antigen. In Caucasians, the antigen frequency of K is about 7%. Due to the strong immunogenicity of K antigen, Koell serves as a test item for blood group detection before blood transfusion. In Chinese population, Kell's blood group has been regarded as a single, basic k, and K frequency is about 0. 07%, so it is not necessary to set the K antigen before transfusion. So far, there are few Kell blood types in China. We divided the KEL gene into individuals of Chinese population, K, k and K _ 0. In this study, 19 exons of KEL gene, combined with sequencing and cloning and sequencing, were used to analyze the bases of these three phenotypes. As a result of the structure, the KEL gene of all K individuals detected was a mutation of exon 6, C698T, resulting in Met193H11 amino acid Change of the KEL gene of an individual and the wild-type gene sequence A total of 87, 665 patients in Shanghai were screened, and 2 cases of K _ 2 were found. The KEL gene of 2 patients with K _ 0 was the same. Change type. The 3rd exon n185 position on one chromosome has a T insertion, so that the original TCT becomes TTC, resulting in Ser62Phe amino group. The acid changes. Also, because insertion T shifts the reading frame, the stop codon appears in advance at exon 4; the other chromosome, exon 7, n715 has a G/ T mutation, causing GAA to become TAAA, and the amino acid occurs Glu239Stop Changes in the KEL gene. The structure was studied. It was found that the K-phenotype and K-phenotype individuals had a complete KEL gene transcript in the web-dyed red blood cells. Different types of transcripts. However, K _ 0 individuals lack complete KEL gene transcripts, and alternative splicing patterns are used in RNA splicing, including partial introns retention and some The transmutation of some exons. In white-lineage cells, the transcripts of all individuals are more than one, spliced by different exons or introns by selective splicing, and KEL gene is woven with K _ 0 individuals. In this case, flow cytometry was used to analyze the expression of Kell antigen on red blood cells and white blood cells. The results showed that there was a strong Kell antigen expression on the red blood cells with K + or k + phenotype, and the red blood cells of K _ 0 individuals did not Expression of any Kell antigen. In white cells, K _ 0 individuals also No Kell proteins were expressed. However, 4 cases of k-individuals had different expression of Kell antigen in different degrees, and 4 cases were used. This was confirmed by different antibodies. Further studies have found that the white cells with Kell antigen expression are mainly CD4 + T cells, CD19 + B cells, and CD8 + T cells, CD56 + NK cell groups. There is no Kell antigen expression. The study of K _ 0 family shows that the first parents' Kell phenotype is normal. and each has a mutant KEL gene. The mutated gene of a chromosome is derived from their parents, respectively. The older sister is genetically inherited from the mutated gene of his mother, and the other chromosome is provided with The wild-type KEL gene is derived from his father. So she has a normal Kell phenotype. Her brother's inheritance is normal. Chromosomes, having a normal Kell phenotype, are genetically engineered due to two different mutations of the KEL gene First of all, a rare K _ 0 phenotype was created and only the KEL gene on one chromosome Mutation does not cause the Kell phenotype to be abnormal K/ k typing kit was developed and applied. A specific primer was designed to establish a sequence-specific primer PCR (PCR-SSP) method for the identification of K and k genotypes. The K/ k PCR-SSP genotyping kit was successfully applied to the screening of K/ k genotype and 2 ISBT (International Society Blood Transfusion).
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R394
本文编号：2299518
[Abstract]:The Kell blood type system is the most important blood type system following ABO and Rh blood type systems. The Kell blood group system has a complex polymorphism. So far, 27 antigens have been found, all of which are caused by point mutation. If all Kell antigens are not expressed, this special phenotype is referred to as K-null (K _ 0). The K antigen corresponding to the wild type k is a low frequency antigen. In Caucasians, the antigen frequency of K is about 7%. Due to the strong immunogenicity of K antigen, Koell serves as a test item for blood group detection before blood transfusion. In Chinese population, Kell's blood group has been regarded as a single, basic k, and K frequency is about 0. 07%, so it is not necessary to set the K antigen before transfusion. So far, there are few Kell blood types in China. We divided the KEL gene into individuals of Chinese population, K, k and K _ 0. In this study, 19 exons of KEL gene, combined with sequencing and cloning and sequencing, were used to analyze the bases of these three phenotypes. As a result of the structure, the KEL gene of all K individuals detected was a mutation of exon 6, C698T, resulting in Met193H11 amino acid Change of the KEL gene of an individual and the wild-type gene sequence A total of 87, 665 patients in Shanghai were screened, and 2 cases of K _ 2 were found. The KEL gene of 2 patients with K _ 0 was the same. Change type. The 3rd exon n185 position on one chromosome has a T insertion, so that the original TCT becomes TTC, resulting in Ser62Phe amino group. The acid changes. Also, because insertion T shifts the reading frame, the stop codon appears in advance at exon 4; the other chromosome, exon 7, n715 has a G/ T mutation, causing GAA to become TAAA, and the amino acid occurs Glu239Stop Changes in the KEL gene. The structure was studied. It was found that the K-phenotype and K-phenotype individuals had a complete KEL gene transcript in the web-dyed red blood cells. Different types of transcripts. However, K _ 0 individuals lack complete KEL gene transcripts, and alternative splicing patterns are used in RNA splicing, including partial introns retention and some The transmutation of some exons. In white-lineage cells, the transcripts of all individuals are more than one, spliced by different exons or introns by selective splicing, and KEL gene is woven with K _ 0 individuals. In this case, flow cytometry was used to analyze the expression of Kell antigen on red blood cells and white blood cells. The results showed that there was a strong Kell antigen expression on the red blood cells with K + or k + phenotype, and the red blood cells of K _ 0 individuals did not Expression of any Kell antigen. In white cells, K _ 0 individuals also No Kell proteins were expressed. However, 4 cases of k-individuals had different expression of Kell antigen in different degrees, and 4 cases were used. This was confirmed by different antibodies. Further studies have found that the white cells with Kell antigen expression are mainly CD4 + T cells, CD19 + B cells, and CD8 + T cells, CD56 + NK cell groups. There is no Kell antigen expression. The study of K _ 0 family shows that the first parents' Kell phenotype is normal. and each has a mutant KEL gene. The mutated gene of a chromosome is derived from their parents, respectively. The older sister is genetically inherited from the mutated gene of his mother, and the other chromosome is provided with The wild-type KEL gene is derived from his father. So she has a normal Kell phenotype. Her brother's inheritance is normal. Chromosomes, having a normal Kell phenotype, are genetically engineered due to two different mutations of the KEL gene First of all, a rare K _ 0 phenotype was created and only the KEL gene on one chromosome Mutation does not cause the Kell phenotype to be abnormal K/ k typing kit was developed and applied. A specific primer was designed to establish a sequence-specific primer PCR (PCR-SSP) method for the identification of K and k genotypes. The K/ k PCR-SSP genotyping kit was successfully applied to the screening of K/ k genotype and 2 ISBT (International Society Blood Transfusion).
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R394
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1 王玲玲;Kell糖蛋白基因结构与多态性研究[D];华东师范大学;2008年
，本文编号：2299518
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