【作者】 张玉娴；

【导师】 朱自严； 杨颖；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 Kell血型系统是非常复杂的血型系统，对白种人来说，在临床上的重要性仅次于ABO血型系统和Rh血型系统。该血型系统已发现27种抗原，其中第8外显子就有5个点突变，编码9种抗原，即Kp^a／Kp^b／Kp^c、KEL11／KEL17、RAZ、VLAN、KTIM、还有一种未命名的新抗原；第9外显子有2个点突变，编码2种抗原，即KEL13、K22；不过目前还没有发现第7外显子是否发生点突变，是否编码新的抗原。这些抗原中有的能引起严重的输血反应和新生儿溶血病（HDN）。KEL基因在分子水平上具有高度多态性，且一般特征是单个碱基发生改变导致单个氨基酸发生改变，导致抗原表达发生改变。在本课题中，随机选取上海市血液中心2006年3月至2007年1月采集的正常人的血样1015例，其中都是汉族人；另外还有2006年5月新疆地区献血员的随机血样55例作为研究对象，对这些样品的KEL基因外显子7～9的多态性作研究。其中这些样品的血液常规检测发现ABO血型正反定型无异常。因为缺乏相应抗体，无法用血清学进行初筛，且没有阳性标本作对照，所以本实验的实验线路是建立能具有大规模筛查能力的PCR-RF-SSCP方法研究KEL基因外显子7～9。首先，扩增150份已知是正常样品的KEL基因外显子7～9；然后用PvuⅡ酶将PCR扩增产物酶切成两部份，以提高结果的分辩度和灵敏度；再用加热变性、骤冷、聚丙烯酰胺凝胶电泳、银染对这些产物形成的单链构象的格局进行分析。结果发现这些正常样品有两种正常格局，这与PCR-SSCP的试验特征有关。随后以已知正常格局以及正常样品为对照，研究1070例随机样品，研究步骤与已知是正常样品的研究步骤一致，将这1070例的样品的格局与正常格局比较一致，挑选出异常格局的样品对其进行测序分析。结果发现这1070份样品中有3个样品具有异常格局，经多次双向测序结果证实，一个样品Exon9 nt1080 G→A突变，该突变是同义突变，不改变氨基酸，将这碱基突变与GeneBank中的序列比对，证实为一新KEL等位基因；第二个样品在内含子7的67位有个C→A的突变，是新的SNP；第三个样品在Exon7发生nt835 G→T突变，该突变使正常的编码谷氨酸的密码子变成终止密码子，导致终止密码子提前成熟。将这三个碱基突变结果提交到GeneBank，获得三个基因序列号，分别是：DQ905957、DQ923321和EF208901。随后对Exon7 nt835 G→T发生突变导致正常的编码谷氨酸的密码子变成终止密码子的先证者及其家系作一详细的血清学和分子生物学分析。通过血清学分析技术、RT-PCR、常规PCR、直接测序及克隆测序等方法对其分子基础作了深入了解。结果发现该样品的血清学结果表现出K₀表型，除了Exon7 nt835位G→T的杂合突变导致编码谷氨酸的密码子变成终止密码子外，在Exon3 nt304位插入了一个T，导致在外显子4里形成一个终止密码子，终止密码子提前成熟；K₀先证者的父亲在Exon7 nt835位有相同的G→T的杂合突变，其血清学结果表现为Kell抗原正常；K₀先证者的母亲在Exon3 nt304位具有相同的T插入；K₀先证者分别遗传了父母亲的无效的KEL等位基因。正是这两个杂合型的无效KEL等位基因导致K₀先证者红细胞上不表达任何Kell抗原。RT-PCR及cDNA测序结果显示K₀先证者只有一个转录本，且该转录本缺失第3外显子，本试验未检测到第7外显子发生碱基改变的转录本，该碱基突变引起终止密码子提前成熟可能由于无义介导的mRNA衰退而易被快速的降解，从而难以被检测到；也不能排除是由于能检测到的转录本为优势表达，阻挡了含第7外显子突变的转录本的检测，这需要进一步的研究。K₀表现型非常罕见，其分子基础有的是纯合无效突变，有的是杂合无效突变，本文的K₀表现型的分子基础是杂合型的无效等位基因。外显子3的T的插入基因结果提交GeneBank，获得基因序列号，即：EF208900。更多还原

【Abstract】 Kell blood group is a complicated system and important in clinical medicine,behind of ABO and Rh blood group. It has displayed 27 antigens,with 5 point mutations in exon 8,coding 9 antigens, Kpa/Kpb/Kpc、KEL11/KEL17、RAZ、 VLAN、 KTIM and one new unnamed antigen, and 2 point mutations in exon9,coding 2 antigens, KEL13、 K22.Currently no point mutation in exon 7 has been reported. Some antigens could cause sever transfusion reaction and hemolytic disease in newborn infants （HDN）. KEL gene is highly polymorphic in molecular basis and mainly single base mutation causes amino acid change,leading to antigen expression change.In this study,blood samples were collected from 1015 unrelated healthy Han nationality random donors in Shanghai Blood Center between March 2006 to January 2007 and other 55 samples from Xinjiang in May 2006.PCR-RF-SSCP was set up and used to screen in large-scale with serology reagents and positive sample unavailable. Firstly, KEL, gene exons 7～9 of these samples were amplified,among them 150 samples were with known normal KEL sequence;Secondly, PCR products were digested with PvuII enzyme;Thirdly,heating to denature and suddenly cooling to form single strand conformation、polyacrylamide electrophoresis followed with silver dying utilized to screen different single strand conformation patterns. Any sample with abnormal or undiscernable patterns would be sequenced further.3 samples were found with unique pattern,their sequencing results showed that one had a nt1080 G→A synonymous mutation in exon9;the second had a C→A mutation in intron7;the last one had a nt835 G→T mutation in exon7, which changed Glu codon to the termination codon,generating a premature termination codon.These 3 new mutations had got the GenBank accession number, DQ905957、DQ923321 and EF208901.For further study of this individual with nt835 G→T mutation in exon7 and her family ,seology、 RT-PCR、 PCR、cloning and sequencing were used to explore molecular basis of this mutaion. Seology showed that the individual was K₀ phenotype,but Kell antigens were normally expressed in her father and mother’s red blood cells.Beside nt835 G→T mutation, K₀ individual also had a T insertion mutation at nt304 in exon3,bringing premature termination codon in exon4, with the GenBank accession number,EF208900.These two heterozygous novel mutaions of the KEL gene may explain the K₀ phenotype. The family study showed the mother had the same T insertion at nt304 in exon3 and the father had a same nt835 G→T mutation in exon7. The results of RT-PCR and cDNA sequencing suggested that K₀ individual had one transcript, which was with the deletion of total exon3. The candidate transcript with mutation in exon7 was not found even with repeated experiments.Generally,mRNAs containing premature termination codons are often thought to be rapidly degraded by nonsense-mediated mRNA decay. Transcript with mutation in exon7 maybe degraded so rapidly that we could not discovered it. But the possibility that this candidate transcript with premature termination codon in exon7 was shielded by the prevailing transcript of exon3 deleton couldn’t be ruled out.更多还原