Kell糖蛋白基因結(jié)構(gòu)與多態(tài)性研究
發(fā)布時(shí)間：2018-10-30 08:44
【摘要】： Kell血型系統(tǒng)是繼ABO,Rh血型系統(tǒng)之后最為重要的血型系統(tǒng)。Kell血型系統(tǒng)具有復(fù)雜的多態(tài)性,到目前為止,共發(fā)現(xiàn)了27種抗原,這27種抗原皆由點(diǎn)突變?cè)斐�。如果所有Kell抗原都不表達(dá),這種特殊的表型稱為K-null(K_0)。與野生型k相對(duì)應(yīng)的K抗原是低頻率抗原。白種人中,K的抗原頻率約為7%。由于K抗原具有較強(qiáng)的免疫原性,所以歐美國家都將Kell作為輸血前血型檢測(cè)的必檢項(xiàng)目。中國人群中,Kell血型一直被認(rèn)為多態(tài)性較單一,基本都為k,且K的頻率約為0.07%,因此輸血前不進(jìn)行K抗原的定型。至今,國內(nèi)也很少有Kell血型的相關(guān)研究。 我們對(duì)中國人群中,K,k,K_0表型的個(gè)體進(jìn)行KEL基因分型研究。分段擴(kuò)增KEL基因19個(gè)外顯子,結(jié)合測(cè)序和克隆測(cè)序的方法,分析上述3種表型的基因結(jié)構(gòu)。結(jié)果發(fā)現(xiàn),檢測(cè)的所有K個(gè)體的KEL基因均為第6外顯子C698T的突變,從而導(dǎo)致Met193Thr氨基酸的改變。k個(gè)體的KEL基因與野生型基因序列一致。篩選了87,665例上海地區(qū)O型獻(xiàn)血員,發(fā)現(xiàn)2例K_0個(gè)體。這2例K_0個(gè)體的KEL基因具有相同的突變類型。一條染色體上第3外顯子nt185位具有一個(gè)T插入,使得原本的TCT變成TTC,導(dǎo)致Ser62Phe氨基酸改變。并且,因?yàn)椴迦隩使閱讀框移位,終止密碼子在第4外顯子提前出現(xiàn);另外一條染色體,第7外顯子nt715位具有G→T突變,使GAA變成TAA,氨基酸發(fā)生Glu239Stop的變化。 我們進(jìn)一步對(duì)KEL基因的轉(zhuǎn)錄本結(jié)構(gòu)進(jìn)行了研究。發(fā)現(xiàn)在網(wǎng)織紅細(xì)胞中,k表型以及K表型的個(gè)體都具有一條完整的KEL基因轉(zhuǎn)錄本,K表型個(gè)體可檢測(cè)到具有突變類型的轉(zhuǎn)錄本。但是K_0個(gè)體缺乏完整的KEL基因轉(zhuǎn)錄本,RNA剪接時(shí)采用選擇性剪接的模式,包括部分內(nèi)含子的保留和某些外顯子的跳躍。白系細(xì)胞中,所有個(gè)體的轉(zhuǎn)錄本都不止一種,由不同的外顯子或內(nèi)含子通過選擇性剪接拼接而成,與K_0個(gè)體網(wǎng)織紅細(xì)胞KEL基因轉(zhuǎn)錄本情況類似。 運(yùn)用流式細(xì)胞技術(shù),分析紅細(xì)胞和白細(xì)胞上Kell抗原的表達(dá)情況,并區(qū)分白系細(xì)胞種類,以了解Kell抗原在T淋巴細(xì)胞,B淋巴細(xì)胞以及NK細(xì)胞上的表達(dá)情況。流式檢測(cè)得到結(jié)果表明,具有K+或k+表型個(gè)體紅細(xì)胞上均具有很強(qiáng)的Kell抗原表達(dá);而K_0個(gè)體的紅細(xì)胞上沒有任何Kell抗原的表達(dá)。在白系細(xì)胞上,K_0個(gè)體同樣不表達(dá)任何Kell蛋白。但4例k個(gè)體各自有不同程度微弱的Kell抗原表達(dá);用4種不同抗體都證實(shí)了這一點(diǎn)。進(jìn)一步的研究發(fā)現(xiàn),具有Kell抗原表達(dá)的白系細(xì)胞主要為CD4+的T細(xì)胞、CD19+的B細(xì)胞,而CD8+T細(xì)胞、CD56+NK細(xì)胞基本沒有Kell抗原表達(dá)。 對(duì)K_0家系的研究證明,先證者的父母Kell表型都正常,各自具有一個(gè)突變的KEL基因。造成先證者K_0表型的兩條染色體突變基因分別來自其父母。先證者的姐姐遺傳得到其母親的突變基因,另外一條染色體上帶有野生型的KEL基因,來自其父親。因此,她具有正常的Kell表型。先證者的弟弟遺傳得到父母正常的染色體,有正常的Kell表型。由于兩種不同突變的KEL基因同時(shí)遺傳給先證者,造成了罕見的K_0表型。而僅僅一條染色體上的KEL基因突變并不能造成Kell表型異常。 K/k分型試劑盒的研制及應(yīng)用。設(shè)計(jì)特異性引物,建立序列特異性引物PCR(sequence-specific primer polymerase chain reaction,PCR-SSP)的方法,用于鑒定K及k基因型,并結(jié)合測(cè)序,鑒定試劑盒的可靠性和重復(fù)性。該K/k PCR-SSP基因分型試劑盒成功應(yīng)用于外籍孕婦產(chǎn)前檢查的K/k基因型篩選和2例ISBT(International Society Blood Transfusion)血型基因定型workshop標(biāo)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).
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R394
本文編號(hào)：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).
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R394
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 王玲玲;Kell糖蛋白基因結(jié)構(gòu)與多態(tài)性研究[D];華東師范大學(xué);2008年
，本文編號(hào)：2299518
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