CN114561458B - 一种与abo血型系统中b变异型相关的snp位点及其应用 - Google Patents
一种与abo血型系统中b变异型相关的snp位点及其应用 Download PDFInfo
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Abstract
本发明的目的是提供一种用于检测ABO血型系统B型变异型的SNP位点,新的B型变异型基因编码区域由起始密码子起第125位插入T碱基。本发明提供了一种新的B型变异型基因,从而完善了ABO基因库,提供了一种快速基因诊断、基因筛查及遗传咨询的途径,应用效果表明本发明所提供的基因的SNP位点及检测引物可以有效的应用于献血员和临床患者外周血进行新的B型变异型基因突变位点的快速检测。
Description
技术领域
本发明属于基因诊断制品技术领域,具体涉及一种与ABO血型系统中B变异型相关的SNP位点及其应用。
发明背景
ABO血型系统是人类认识的第一个血型系统,也是目前为止最为重要的血型系统,该血型系统在临床输血和新生儿溶血病中占有重要的地位。根据红细胞表面特异性抗原(凝集原)A或/和特异性抗原B的分布,人们把红细胞分为A、B、AB和O四型,这就组成了ABO血型系统。进一步,随着分子生物学的发展,F.Bernstein证明ABO血型分别由三个复等位基因控制。ABO抗原的前体是H抗原,由FUT1基因编码;而A基因编码一种叫N-乙酰氨基半乳糖基转移酶的蛋白质(A酶),能把H抗原转化成A抗原;B基因编码一种叫半乳糖基转移酶的蛋白质(B酶),能把H抗原转化成B抗原;O基因不能编码有活性的酶,故只有H抗原。若A基因或B基因发生突变,就会影响所编码转移酶的活性,导致将H抗原转变为A抗原或B抗原的能力降低,从而在血清学实验中表现为弱A或弱B表型。
目前对ABO血型的鉴定基本上是采用血清学实验方法,研究人员通过调整试剂抗-A和抗-B的效价,使得临床检验科室可以在日常血型血清学鉴定中发现弱A和/或弱B表型。但血型血清学检测方法会受到疾病、药物或者个体的不规则抗体的干扰,比如肿瘤、白血病等某些疾病会影响血型抗原从而导致正定型不准确,而血清中杂蛋白、自身抗体或不规则抗体的存在则会干扰反定型,这些都会导致定型困难甚至错误。
近年来,ABO基因分型技术日趋发展成熟,而基因检测可以克服血清学检测中的干扰因素,为临床解决了一些疑难血型鉴定问题,目前已是一种比较独立简便的分型方法,并且得到了广泛应用。而随着基因分型技术的普及,越来越多的SNP位点被发掘并被认可,这不仅降低了临床发生ABO定型错误的风险,而且扩大了人们对ABO亚型的认可度,同时又进一步促进了分型技术的发展,因此,SNP位点的充分发掘和完善对临床血型鉴定和安全输血提供了保障,并且对基因分型技术的广泛应用奠定了基础。
发明内容
本发明的目的是提供与ABO血型系统中B变异型相关的SNP位点及其应用,可以完善ABO基因库的数据,从而弥补现有技术的不足。
申请人对一个呈常染色体9q34.1-q34.2遗传的变异型血型基因家系成员进行测序,找到了该家系成员B型变异型基因存在遗传上的突变,造成了B抗原数量和质量的改变。
本发明首先提供一种与ABO血型系统中B变异型相关的SNP位点,为ABO血型B等位基因(NCBI Reference Sequence:MZ466383.1)编码区ATG起始第125位插入T碱基,即核苷酸序列为SEQ ID NO:1所述片段的第125。
ATGGCCGAGGTGTTGCGGACGCTGGCCGGAAAACCAAAATGCCACGCACTTCGACCTATGATCCTTTTCCTAATAATGCTTGTCTTGGTCTTGTTTGGTTACGGGGTCCTAAGCCCCAGAAGTCTTAATGCCAGGAAGCCTGGAACGGGGGTTCTGCATGGCTGTTAGGGAACCTGACCATCTGCAGCGCGTCTCGTTGCCAAGGATGGTCTACCCCCAGCCAAAGGTGCTGACACCGTGTAGGAAGGATGTCCTCGTGGTGACCCCTTGGCTGGCTCCCATTGTCTGGGAGGGCACGTTCAACATCGACATCCTCAACGAGCAGTTCAGGCTCCAGAACACCACCATTGGGTTAACTGTGTTTGCCATCAAGAAATACGTGGCTTTCCTGAAGCTGTTCCTGGAGACGGCGGAGAAGCACTTCATGGTGGGCCACCGTGTCCACTACTATGTCTTCACCGACCAGCCGGCCGCGGTGCCCCGCGTGACGCTGGGGACCGGTCGGCAGCTGTCAGTGCTGGAGGTGGGCGCCTACAAGTGCTGGCAGGACGTGTCCATGCGCCGCATGGAGATGATCAGTGACTTCTGCGAGCGGCGCTTCCTCAGCGAGGTGGATTACCTGGTGTGCGTGGACGTGGACATGGAGTTCCGCGACCATGTGGGCGTGGAGATCCTGACTCCGCTGTTCGGCACCCTGCACCCCAGCTTCTACGGAAGCAGCCGGGAGGCCTTCACCTACGAGCGCCGGCCCCAGTCCCAGGCCTACATCCCCAAGGACGAGGGCGATTTCTACTACATGGGGGCGTTCTTCGGGGGGTCGGTGCAAGAGGTGCAGCGGCTCACCAGGGCCTGCCACCAGGCCATGATGGTCGACCAGGCCAACGGCATCGAGGCCGTGTGGCACGACGAGAGCCACCTGAACAAGTACCTACTGCGCCACAAACCCACCAAGGTGCTCTCCCCCGAGTACTTGTGGGACCAGCAGCTGCTGGGCTGGCCCGCCGTCCTGAGGAAGCTGAGGTTCACTGCGGTGCCCAAGAACCACCAGGCGGTCCGGAACCCGTGA(SEQ ID NO:1)。
本发明的另一个方面还提供检测上述的SNP位点的制剂在制备用于检测红细胞ABO血型的制品中的应用,
所述的制剂,作为一个实施例的具体记载,为PCR扩增测序引物;
所述的制品为PCR扩增测序试剂盒,其中包含有检测上述SNP位点的直接扩增引物;
其中扩增引物的序列信息如下:
ABO-3F:5'-TTGCTGAGAACTTGCCCTACC-3'(SEQ ID NO:2)、
ABO-3R:5'-GACCCTTCAAGGCCACAGAG-3'(SEQ ID NO:3)。
本发明通过检测筛选获得的SNP位点,从而提供了一种有效的ABO血型基因诊断途径,完善了ABO基因数据库,应用效果表明本发明所提供的基因SNP位点及检测引物可以有效的用于临床患者外周血进行ABO血型B型变异型基因突变位点的快速检测。
附图说明
图1先证者ABO基因Exon3 PCR产物直接测序结果图,采用ABO-3F和ABO-3R引物进行PCR扩增,采用ABO-3R引物对PCR产物进行测序,黑色箭头指向双峰起始位点,提示其中一条链有碱基插入或缺失,经分析应为第125位(ATG起始)插入T碱基。
图2先证者ABO基因Exon3克隆测序结果图,将PCR产物进行克隆,挑取单克隆测序,测序结果与ABO*B.01的Exon3(第一行碱基)进行序列分析比较,黑色箭头指向先证者Exon3的3个克隆中存在T碱基插入,结合直接测序结果,证实B基因在125bp(从ATG起始)插入T碱基。
具体实施方式
ABO基因位于染色体9q34.1-q34.2,可转录成1065bp的mRNA,直接翻译形成355个氨基酸组成的蛋白质。其中ABO*B.01(NCBI登录号为MZ466383.1)编码半乳糖转移酶的蛋白质(B酶),负责B抗原的形成。
对于本发明所涉及SNP标记(single nucleotide polymorphism,SNP,即单核苷酸多态性)是指基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。SNP表现出的多态性仅涉及到单个碱基的变异,表现是有转换、颠换、插入和缺失等。
申请人在一个ABO血型B亚型家系中进行DNA测序时,发现了会导致B型变异型基因的SNP突变。
下面结合实施例对本发明进行详细的描述。
实施例1:SNP标记的筛选
1、提取外周血基因组DNA:
在符合国家相关政策规定,并在取样对象同意的基础上,抽取B型变异型基因家系成员外周静脉血2-5mL,放入EDTA-Na2抗凝管内,-80℃冻存备用;DNA提取使用LIFE公司的DNA提取试剂盒,具体操作流程如下:
冻存的EDTA抗凝血在室温融化后,取500μL放于离心管,加入等体积的红细胞裂解液(pH 8.0),涡旋混匀5分钟,12000rpm离心5分钟,弃上清。重复加入红细胞裂解液500μL涡旋混匀5分钟,12000rpm离心5分钟,弃上清。
沉淀物涡旋混匀5分钟,加入核裂解液50μL,涡旋混匀5分钟,置于56℃金属浴15分钟。从水浴中取出样品,加入蛋白清除液135μL。涡旋充分混匀,至于4℃30分钟。
室温下12000rpm离心5分钟,吸取上清液(约200μl)至一新离心管中。加入冰乙醇500μL,反复震荡离心管,直至白色DNA沉淀物析出。室温12000rpm离心2分钟,弃上清。向DNA沉淀加入75%乙醇,漂洗一次,室温12000rpm离心3分钟,弃上清,置于室温中挥发剩余乙醇,最后加入50μL TE(pH8.0),4℃过夜溶解DNA。
对提取的DNA行琼脂糖胶电泳,并应用紫外分光光度计在260nm和280nm比色,检测DNA纯度及浓度。
2、直接测序法寻找先证者B变异型基因的突变位点
PCR扩增目的片段:反应条件与反应体系:
(1)PCR反应条件:95℃预变性10分钟;94℃60秒,63℃90秒,72℃60秒,10个循环;94℃60秒,61℃90秒,72℃60秒,25个循环;72℃延伸10min,扩增产物10℃保温。
(2)反应体系:(TAKARA Ex-Taq polymerase)
应用该反应体系进行先证者的基因组DNA模板与上述ABO-3F、ABO-3R引物的扩增反应。
PCR产物测序:应用常规Sanger测序法对上述PCR产物进行测序,其中应用引物ABO-3R:5'-GACCCTTCAAGGCCACAGAG-3'在先证者发现ABO血型基因第3外显子第125位(ATG起始)起存在双峰(图1),提示该先证者双链DNA中有一条链存在碱基插入或缺失,经分析应为第125位(ATG起始)插入T碱基。多次测序结果表明该突变位点并不是因为扩增或测序错误引进的。
3、对ABO基因第3外显子进行单倍型测序,确定先证者B变异型基因的突变位点;
将上述PCR产物克隆入T载体,挑取多个菌落进行单倍型序列测定,单倍型测序引物使用T载体上M13F-47通用引物(测序引物也可以选用从B型变异基因上设计的,能扩增SNP位点的其它引物)得到单倍型序列,与NCBI参考序列ABO*B.01(MZ466383.1)进行比较,发现在第3外显子第125位插入T碱基(图2),该突变为一新发现突变。该突变不存在于下面的四个数据库中:单核苷酸多态性数据库(ftp://ftp.ncbi.nih.gov/snp/database/),千人基因组计划(ftp://ftp-trace.ncbi.nih.gov/1000genomes/ftp/),Hapmap8数据库(http://hapmap.ncbi.nlm.nih.gov/),及炎黄数据库(http://yh.genomics.org.cn/),表明该突变非常罕见,而在近300例正常当地人群的外周血基因组DNA样本中进行该位点的突变筛查,未发现该突变。
在青岛市中心血站输血医学研究所收集的先证者家系成员,提取他们的外周血基因组DNA,应用上述DNA作为模板与ABO-3F、ABO-3R引物进行PCR目的片段的扩增,常规Sanger测序法应用ABO-3R引物对PCR产物进行直接测序,发现该先证者的2名家系成员的ABO血型B基因存在本发明中发现的SNP突变,即编码区域由起始密码子起第125位插入T碱基。继续对发现SNP突变的2位家系成员进行ABO基因第3外显子的单倍型测序,应用上述提取的2名家系成员的基因组DNA作为模板与ABO-3F、ABO-3R引物进行PCR目的片段的扩增,将PCR产物克隆入T载体,挑取多个菌落进行单倍型序列测定,单倍型测序引物使用M13F-47引物,确定样本的单倍型序列存在起始密码子起第125位插入T碱基。
通过上述的分析,证明该SNP位点能够精准的确定待测者的ABO血型,对于基因确定ABO血型提供了更加准确的数据背景,对患者制定输血政策具有重要意义。
序列表
<110> 青岛市中心血站
<120> 一种与ABO血型系统中B变异型相关的SNP位点及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1066
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggccgagg tgttgcggac gctggccgga aaaccaaaat gccacgcact tcgacctatg 60
atccttttcc taataatgct tgtcttggtc ttgtttggtt acggggtcct aagccccaga 120
agtcttaatg ccaggaagcc tggaacgggg gttctgcatg gctgttaggg aacctgacca 180
tctgcagcgc gtctcgttgc caaggatggt ctacccccag ccaaaggtgc tgacaccgtg 240
taggaaggat gtcctcgtgg tgaccccttg gctggctccc attgtctggg agggcacgtt 300
caacatcgac atcctcaacg agcagttcag gctccagaac accaccattg ggttaactgt 360
gtttgccatc aagaaatacg tggctttcct gaagctgttc ctggagacgg cggagaagca 420
cttcatggtg ggccaccgtg tccactacta tgtcttcacc gaccagccgg ccgcggtgcc 480
ccgcgtgacg ctggggaccg gtcggcagct gtcagtgctg gaggtgggcg cctacaagtg 540
ctggcaggac gtgtccatgc gccgcatgga gatgatcagt gacttctgcg agcggcgctt 600
cctcagcgag gtggattacc tggtgtgcgt ggacgtggac atggagttcc gcgaccatgt 660
gggcgtggag atcctgactc cgctgttcgg caccctgcac cccagcttct acggaagcag 720
ccgggaggcc ttcacctacg agcgccggcc ccagtcccag gcctacatcc ccaaggacga 780
gggcgatttc tactacatgg gggcgttctt cggggggtcg gtgcaagagg tgcagcggct 840
caccagggcc tgccaccagg ccatgatggt cgaccaggcc aacggcatcg aggccgtgtg 900
gcacgacgag agccacctga acaagtacct actgcgccac aaacccacca aggtgctctc 960
ccccgagtac ttgtgggacc agcagctgct gggctggccc gccgtcctga ggaagctgag 1020
gttcactgcg gtgcccaaga accaccaggc ggtccggaac ccgtga 1066
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ttgctgagaa cttgccctac c 21
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gacccttcaa ggccacagag 20
Claims (2)
1.一种检测SNP位点的PCR扩增测序引物在制备用于检测红细胞ABO血型的制品中的应用;所述的SNP位点是核苷酸序列为SEQ ID NO:1的ABO血型B等位基因编码区ATG起始第125位插入T碱基。
2.如权利要求1所述的应用,其特征在于,所述的检测红细胞ABO血型的制品为PCR扩增测序试剂盒。
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