【摘要】： 研究背景 ABO血型系統(tǒng)是人類最先發(fā)現(xiàn)的重要血型系統(tǒng),在輸血醫(yī)學和器官移植等方面具有重要意義。經(jīng)典的血型血清學方法按照抗原的不同可分為A、B、O、AB四種表型,此外ABO血型系統(tǒng)也存在一定的亞型(弱A或弱B)。上世紀九十年代明確了ABO血型基因的核苷酸序列、結構以及一些常見突變點。ABO基因位于染色體9q341-34.2,包含長度大小從28-688bp不等的7個外顯子�，F(xiàn)一般將A101作為參考序列,B101有7個核苷酸與A101基因不同,導致蛋白上四個氨基酸變化,其中兩處替換(L266M和G268A)決定酶的特性。常見的O等位基因上為第6外顯子第261位單核苷酸缺失,造成酶活力的喪失。此外也發(fā)現(xiàn)少量的261位未缺失的O等位基因。隨著分子技術不斷完善,現(xiàn)ABO血型基因分型的方法有PCR-RFLP、PCR-SSP、PCR-SSCP、PCR-SSO、PCR-SBT和PCR-GeneScan等,其中最佳的方法是PCR-SBT。到目前為止,通過分子生物學方法已鑒定200多個ABO等位基因。 本研究的目的在于建立ABO血型基因診斷的測序方法(PCRSBT),獲取浙江漢族人群ABO血型基因多態(tài)性的分布資料,并分析獻血人群ABO亞型和不規(guī)則抗體分布情況,為臨床輸血提供更好的技術指導和幫助。實驗使用PCR-SBT方法對ABO基因第6、7外顯子進行了測序分析,并檢測了部分浙江漢族人群標本,發(fā)現(xiàn)了三個新的等位基因。 實驗方法 1、標本來源 1.1 ABO血型表型分析:2008年7-9月來浙江省血液中心的隨機無償獻血者30519名,通過填寫征詢表確認其民族為漢族。 1.2人群中ABO亞型和不規(guī)則抗體調查:2008年來浙江省血液中心的隨機無償獻血者,共計標本數(shù)為167888人次。 1.3 ABO血型等位基因分布情況:標本來自2008年7-9月浙江省血液中心的隨機無償獻血者。所有標本經(jīng)其知情同意后采集5ml EDTA抗凝血,分別吸取1-2ml血樣貯存在-20℃以下備用。標本總數(shù)為417例。 2、血清學方法測定 2.1 ABO表型檢測:采用PK7200全自動血型檢測系統(tǒng)測定ABO血清學表型,參照儀器說明書進行操作。 2.2 ABO亞型鑒定:對于正反定型不一致或者疑是亞型的標本進行鑒定,采用試管法,具體參照中心參比實驗有關操作說明進行。 2.3不規(guī)則抗體鑒定采用凝集技術在PK7200自動血型檢測系統(tǒng)檢測。 3、標本DNA提取:采用QIAamp Blood Kit商用抽提試劑盒。 4、ABO基因第6-7外顯子測序分析 4.1 PCR擴增:PCR擴增引物序列參照ABO基因序列,采用一對引物對進行5-7外顯子擴增,PCR引物序列和擴增條件參照正文,擴增反應體系總體積為25μl。 4.2 PCR產(chǎn)物純化:每孔中加入核酸外切酶Ⅰ1μl(5U),蝦堿性磷酸酶2μl(2U),ABI公司的9700型PCR自動擴增儀進行酶切反應,37℃60min,80℃15min。 4.3測序反應:分別針對外顯子6、7設計四個測序引物,以純化PCR產(chǎn)物為模板按BigDye Sequencing kit(ABI公司)試劑盒操作進行測序反應,ABI公司9700型PCR自動擴增儀擴增,擴增條件為95℃預變性5min,95℃10s,50℃10s,60℃4min,25個循環(huán),冷卻至4℃。 4.4測序反應純化和電泳分析:采用乙醇/醋酸鈉法純化,然后在ABI 3730測序儀上進行測序電泳分析。 4.5測序結果分析和判斷:SeqScape V2.5軟件進行數(shù)據(jù)分析。 5磁珠M-270鏈親和素方法分離新等位基因的單倍體 當鑒定的新等位基因為雜合子,采用磁珠M-270鏈親和素方法(Invitrogen,Wisconsin,USA)分離其單倍體。主要分兩步,第一步模板DNA與5′端生物素標記特異性探針反應,生物素探針只與特定的DNA單鏈結合。第二步將生物素標記單鏈DNA加入鏈親和素化磁珠M-270形成生物素標記DNA-鏈親和素包被磁珠,經(jīng)洗滌、磁選后得到的DNA單鏈可作為ABO基因外顯子6-7的擴增模板。 6、統(tǒng)計學分析:人群基因頻率(gene frequency,GF)采用直接計數(shù)法計算,即檢測出的基因個數(shù)/2N(N=標本數(shù))。Hardy-Weinberg(H-W)平衡檢驗采用x~2檢驗。p＜0.05表示具有統(tǒng)計學意義。 結果 1、30519例浙江漢族人群ABO血型表型分布情況 采用凝集方法,結果顯示A型為9274例,占30.4%。B型為7986例,占26.2%。O型為10542例,占34.5%。AB型為2717例,占8.9%。ABO血型表型分布中O＞A＞B＞AB。 2、ABO亞型與不規(guī)則抗體檢測情況 從167888例標本中檢出8例ABO亞型,ABO亞型頻率為4.8/10萬。檢出43例不規(guī)則抗體,不規(guī)則抗體檢出頻率為2.6/萬。其中抗M為32例,占74.4%�？笵為4例,占9.3%。冷凝集為3例,占7.0%�？笻、抗C、抗P1、自身抗體各為1例。抗M為最常見的不規(guī)則抗體。 3、浙江漢族人群ABO等位基因型分布情況 結果顯示外顯子6、7序列確定的ABO基因型與表型完全相符。在浙江漢族人群中共發(fā)現(xiàn)27種基因型,頻率大于1%的為11種。常見的基因型分布為A102/O01為54例(12.95%),A102/O02為34例(8.15%),A102/A102為19例(4.56%);B101/O01為56例(13.43%),B101/O02為38例(9.11%),B101/B101為20例(4.80%),O01/O02為70例(16.78%),O01/O01為44例(10.55%),O02/O02為19例(4.56%),A102/B101為33例(7.91%)、人群中基因型為純合子的為102例(24.46%),雜合子為315例(75.54%)。 4、浙江漢族人群ABO等位基因分布情況 412例標本中共檢測到14個ABO等位基因,包括五個常見的等位基因:A101、A102、B101、O01和O02。此外有6個較少見的等位基因:A205、B110、O04、O05、O07、O50,還有3個新的等位基因:B112、B526C和O743C。頻率大于1%的為A101(1.56%)、A102(19.78%)、B101(20.62%)、O01(33.69%)和O02(22.30%)。人群中最常見的等位基因為O01,在A基因中共發(fā)現(xiàn)3個等位基因A101、A102、A205,A102頻率大于A101,A205發(fā)現(xiàn)有2例占0.240%。在B基因中共發(fā)現(xiàn)4個等位基因B101,B110、B112、B526C。在O基因中共發(fā)現(xiàn)7個等位基因O01、O02、O04、O05、O07、O50、O743C。 5、3個新等位基因情況 在417例標本中發(fā)現(xiàn)3個新的等位基因,三個新等位基因的DNA序列已提交GenBank(序列號為FJ599810、FJ638611和FJ851690)。這三個突變點都發(fā)生在外顯子7區(qū)域。 5.1 B112(559T):在一個B/O雜合子個體中發(fā)現(xiàn)該等位基因(B112/O01),DNA磁珠純化技術分離得到單倍體,測序發(fā)現(xiàn)在B型單鏈上存在突變。B112與B101比較在第559位C＞T,導致一個氨基酸改變(Arg187Cys),血清學上未觀察到血清學特性的變化。 5.2 B526C:在一個AB個體中發(fā)現(xiàn)(A102/B526C),通過磁珠純化獲取單鏈,結果發(fā)現(xiàn)B等位基因在526位G＞C,導致一個氨基酸改變。526位是A和B基因間的差異堿基,在該處A型是C、B型是G。該標本未觀察到任何血清學特性變化。 5.3 0743C:在3個標本中發(fā)現(xiàn)存在0743C等位基因,其中兩個O型雜合子(O01/0743C)和一個B型雜合子(B101/0743C)。與O01序列比較,0743C發(fā)生一個突變743G＞C,由于與O01相同存在261位G缺失,在第261位以后發(fā)生閱讀框架的移動,形成新的終止密碼子,提前終止了轉移酶的翻譯。 結論 1、獲取了浙江漢族人群ABO血型表型分布情況。 2、獲取了浙江漢族人群ABO亞型與不規(guī)則抗體分布情況。 3、首次獲取了浙江漢族人群ABO血型等位基因多態(tài)性數(shù)據(jù)。 4、發(fā)現(xiàn)了三個新的等位基因。
[Abstract]:Research background
ABO blood group system is an important blood group system first discovered by human beings, which is of great significance in blood transfusion medicine and organ transplantation. Classical blood group serology can be divided into four phenotypes A, B, O and AB according to different antigens. In addition, ABO blood group system also has certain subtypes (weak A or weak B). ABO blood group base was defined in the 1990s. ABO gene is located on chromosome 9q341-34.2 and contains seven exons ranging in length from 28 BP to 688 bp. Al101 is generally used as a reference sequence. There are seven nucleotides in B101 that are different from A101, resulting in changes in four amino acids in the protein. Two substitutions (L266M and G268A) determine enzymes. The most common O allele is the 261 single nucleotide deletion at exon 6, resulting in the loss of enzyme activity. A small number of 261 O alleles are also found. With the development of molecular techniques, ABO genotyping methods are PCR-RFLP, PCR-SSP, PCR-SSCP, PCR-SSO, PCR-SBT and PCR-GeneScan, among which the best are PCR-RFLP, PCR-SSP, PCR-SBT and PCR-GeneScan. The method is PCR-SBT.. Up to now, more than 200 ABO alleles have been identified by molecular biology.
The purpose of this study is to establish a sequencing method for ABO genotyping (PCR-SBT), obtain the distribution of ABO genotype gene polymorphism in Zhejiang Han population, and analyze the distribution of ABO subtypes and irregular antibodies in blood donors, so as to provide better technical guidance and help for clinical blood transfusion. Three new alleles were found in some samples of Zhejiang Han population.
Experimental method
1, specimen sources
1.1 ABO blood group phenotype analysis: 30 519 random blood donors from Zhejiang Blood Center from July to September 2008 were confirmed to be Han nationality by filling in a questionnaire.
1.2 Survey of ABO subtypes and irregular antibodies in the population: A total of 167 888 random blood donors from Zhejiang Blood Center in 2008.
1.3 Distribution of ABO blood group alleles: Specimens were collected from random and unpaid donors in Zhejiang Blood Center from July to September 2008. All specimens were collected with their informed consent and 5 ml EDTA anticoagulant, and 1-2 ml of blood samples were collected and stored below - 20 C for reserve. The total number of specimens was 417.
2. Serological method.
2.1 ABO phenotype detection: PK7200 automatic blood group detection system was used to determine ABO serological phenotype, and the operation was carried out according to the instructions of the instrument.
2.2 ABO subtype identification: For samples with inconsistent positive and negative stereotypes or suspected subtypes, test tube method was used, with specific reference to the operation instructions of the central reference experiment.
2.3 the identification of irregular antibodies was detected by agglutination technique in PK7200 automatic blood typing system.
3, specimen DNA extraction: QIAamp Blood Kit commercial extraction kit.
4, sequencing analysis of exon 6-7 of ABO gene.
4.1 PCR amplification: PCR amplification primer sequence refers to ABO gene sequence, using a pair of primers for 5-7 exon amplification, PCR primer sequence and amplification conditions refer to the text, the total volume of the amplification reaction system is 25 mu L.
4.2 PURIFICATION OF PCR PRODUCTS: Each pore was digested with exonuclease_1_ (5U), shrimp alkaline phosphatase 2_ (2U) and ABI company's 9700 PCR automatic amplifier, at 37 60 min and 80 15 min.
4.3 Sequencing reaction: Four primers were designed for exon 6 and exon 7 respectively. The purified PCR products were used as templates for sequencing according to BigDye Sequencing kit (ABI Company) kit. The amplification conditions were pre-denatured at 95 C for 5 minutes, pre-denatured at 95 C for 10 seconds, 10 seconds at 50 C for 4 minutes, and cooled to 4 C for 25 cycles.
4.4 Sequencing reaction purification and electrophoresis analysis: Purification by ethanol/sodium acetate method, and then sequencing electrophoresis analysis on ABI 3730 sequencer.
4.5 analysis and judgement of sequencing results: SeqScape V2.5 software for data analysis.
5 magnetic beads M-270 chain avidin method for isolation of haploid from new alleles
When a new allele was identified as a heterozygote, the haploid was isolated by magnetic bead M-270 chain avidin method (Invitrogen, Wisconsin, USA). The first step was to react the template DNA with a 5'-terminal biotin-labeled specific probe. The biotin probe was only bound to a specific DNA single strand. The second step was to add the biotin-labeled single strand DNA to the chain affinity. The biotin-labeled DNA-chain avidin coated magnetic beads were formed by the biotin-labeled magnetic beads M-270. After washing and magnetic separation, the DNA single strands could be used as amplification templates for exon 6-7 of ABO gene.
6. Statistical analysis: The population gene frequency (GF) was calculated by direct counting method, i.e. the number of detected genes / 2N (N = the number of samples). Hardy-Weinberg (H-W) balance test was performed by X-2 test. P < 0.05 showed statistical significance.
Result
Distribution of ABO blood group phenotypes in 130519 Han population in Zhejiang
Agglutination was used in 9274 cases (30.4%) of type A, 7986 cases (26.2%) of type B, 10542 cases (34.5%) of type O, 2717 cases (8.9%) of type ABO, and O > A > B > AB.
2, the detection of ABO subtypes and irregular antibodies.
ABO subtypes were detected in 8 out of 16788 specimens. The frequency of ABO subtypes was 48/100 000. 43 irregular antibodies were detected. The frequency of irregular antibodies was 26/10 000. Among them, 32 were anti-M, accounting for 74.4%. Anti-D was 4, accounting for 9.3%. Condensation was 3, accounting for 7.0%. Anti-H, anti-C, anti-P1, and autoantibodies were the most common irregular antibodies.
3, the distribution of ABO alleles in Zhejiang Han population.
The results showed that the ABO genotypes determined by exon 6 and exon 7 were completely consistent with the phenotype. Twenty-seven genotypes were found in Zhejiang Han population with frequencies greater than 1%. The common genotypes were A102/O01 in 54 cases (12.95%), A102/O02 in 34 cases (8.15%), A102/A102 in 19 cases (4.56%), B101/O01 in 56 cases (13.43%), B101/O02 in 38 cases (9.11%) and B101/O02 in 38 cases (9.11%). 01/B101 was 20 cases (4.80%), O01/O02 was 70 cases (16.78%), O01/O01 was 44 cases (10.55%), O02/O02 was 19 cases (4.56%), A102/B101 was 33 cases (7.91%), 102 cases (24.46%) were homozygotes and 315 cases (75.54%) were heterozygotes.
4, the distribution of ABO alleles in Zhejiang Han population.
Fourteen ABO alleles were detected in 412 specimens, including five common alleles: A101, A102, B101, O01 and O02. In addition, six rare alleles were found: A205, B110, O04, O05, O07, O50, and three new alleles: B112, B526C and O743C. Frequencies greater than 1% were A101 (1.56%), A102 (19.78%), B101 (20.62%), O01 (33.69%) and O02 (22.3%). The most common allele in the population was O01. Three alleles A101, A102, A205, A102 were found in A gene with frequencies higher than A101, and 2 alleles in A205 accounted for 0.240%. Four alleles B101, B110, B112 and B526C were found in B gene. Seven alleles O01, O02, O04, O05, O07, O50, O743C.
5,3 new alleles
Three new alleles were found in 417 specimens. The DNA sequences of three new alleles were submitted to GenBank (FJ599810, FJ638611 and FJ851690). All three mutations occurred in exon 7 region.
5.1 B112 (559T): The allele (B112/O01) was found in a B/O heterozygote, and the haploid was isolated by magnetic bead purification technique. Mutations were found in the B-type single strand. B112 was compared with B101 at position 559 C > T, resulting in an amino acid change (Arg187Cys). Serological changes were not observed.
5.2 B526C: A single strand was found in an AB individual (A102/B526C) and purified by magnetic beads. It was found that the B allele at position 526 G > C resulted in an amino acid change.
5.3 0743C: Two O-type heterozygotes (O01/0743C) and one B-type heterozygote (B101/0743C) were found in three specimens. Compared with O01 sequence, a mutation 743G > C occurred in 0743C. Because of 261 G deletion, the reading frame shifted after position 261, forming a new termination codon and terminating ahead of time. The translation of the transferase was stopped.
conclusion
1, the distribution of ABO blood group phenotypes in Zhejiang Han population was obtained.
2, we obtained the distribution of ABO subtypes and irregular antibodies in Zhejiang Han population.
3, we obtained the ABO blood group allele polymorphism data of Han nationality in Zhejiang for the first time.
4, three new alleles were found.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R394

【引證文獻】

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1 馮君;應曉君;;浙江漢族人ABO血型與肺癌相關性分析[J];哈爾濱醫(yī)藥;2013年02期

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本文編號：2226973