本文選題：人類血小板抗原　切入點：同種免疫　出處：《福建醫(yī)科大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的建立人類血小板抗原HPA-1～7和-15的基因分型方法同步PCR-SSP,并以該法調(diào)查分析福建省漢族獻血者HPA基因頻率;建立HPA-15基因分型參考DNA細胞系,即B淋巴母細胞系(BLCLs)。 方法(1).檢索HPA基因序列,用Primer Premier 5.0設計HPA引物和內(nèi)參引物(HGHG),每對引物進行BLAST。對HPA各系統(tǒng)和HGHG內(nèi)參作溫度梯度,探索最佳退火溫度,用Touch Down調(diào)節(jié)各HPA系統(tǒng)和HGHG內(nèi)參的反應體系。(2).經(jīng)過優(yōu)化的反應體系對HPA基因型已知的參考DNA標本進行分型,驗證方法的準確性,建立同步PCR-SSP。(3).配制引物混合物并分裝。用同步PCR-SSP調(diào)查160名福建省漢族獻血者的HPA基因頻率,同時驗證該引物混合物的重復性。(4).饑餓培養(yǎng)B95-8細胞至二周左右,收集Epstein-Barr病毒。(5).用PCR-SSP篩選三種HPA-15基因型血標本。EB病毒體外轉(zhuǎn)化血標本B淋巴細胞,建立永生性B淋巴母細胞系(BLCLs)。(6).提取BLCLs基因組DNA,以PCR-SSP和巢式PCR鑒定其HPA-15基因型。鑒定培養(yǎng)3代、10代和15代BLCLs的HPA-15基因型,驗證其基因型的穩(wěn)定性。 結(jié)果(1).建立的同步PCR-SSP操作簡便、快速,準確性和重復性均為100%。(2).福建漢族獻血者HPA-3a和HPA-15a基因頻率較其它HPA系統(tǒng)a基因低,兩者分別為0.5406和0.5531,兩系統(tǒng)的雜合子百分率分別為53.13%和45.63%;HPA-4a和-7a基因頻率大于0.9999(檢測結(jié)果為1.0000),HPA-1、-2、-5、-6系統(tǒng)a基因頻率均大于0.9500,分別為:0.9938、0.9688、0.9813、0.9625。(3).共建成7株BLCLs,選擇4株凍存(HPA-15a/b 2株、HPA-15a/a 1株、HPA-15b/b 1株),BLCLs平均建系時間為八周;實驗證明4株BLCLs建系前后HPA-15基因型完全吻合。(4).培養(yǎng)不同代次(3代、10代、15代)的BLCLs細胞系HPA-15基因型完全一致,從而證實BLCLs作為HPA-15基因分型的參考物質(zhì)穩(wěn)定可靠。(5).三種HPA-15基因型BLCLs細胞系的建立方便了HPA-15基因分型時的質(zhì)量控制,復蘇后的BLCLs僅需培養(yǎng)一周即可提取DNA,結(jié)果準確可靠。 結(jié)論合理設計引物,優(yōu)化循環(huán)參數(shù)和反應體系可以建立HPA基因分型的同步PCR-SSP方法;福建漢族獻血者HPA(1～7和15)的基因頻率與韓國、越南、臺灣等國家和地區(qū)的種族相似,與歐洲、非洲、美洲和澳洲等地區(qū)的種族差異較大;建立HPA-15基因分型的參考DNA細胞系可行。
[Abstract]:Objective to establish a genotyping method for human platelet antigen HPA-1~7 and -15, and to investigate and analyze the frequency of HPA gene in Han blood donors in Fujian province by using this method, and to establish a reference DNA cell line for HPA-15 genotyping, that is, B lymphoblastoid cell line. Methods HPA gene sequence was searched, and HPA primers and internal reference primers were designed with Primer Premier 5.0. Each pair of primers was used for BLAST.The temperature gradient of HPA system and HGHG internal parameters was used to explore the optimum annealing temperature. Touch Down was used to regulate the reaction system of HPA system and HGHG internal parameters. The reference DNA samples with known HPA genotypes were typed by the optimized reaction system, and the accuracy of the method was verified. The HPA gene frequency of 160 Han blood donors in Fujian Province was investigated by synchronous PCR-SSP, and the reproducibility of the primer mixture was verified. The B95-8 cells were cultured for about two weeks after starvation. Epstein-Barr virus was collected. PCR-SSP was used to screen blood samples of three HPA-15 genotypes. Epstein-Barr virus (EBV) was transformed into B lymphocytes from blood samples in vitro. An immortalized B lymphoblastoid mother cell line was established. BLCLs genomic DNA was extracted and its HPA-15 genotypes were identified by PCR-SSP and nested PCR. The HPA-15 genotypes of 10 and 15 generations of BLCLs were identified to verify the stability of their genotypes. Results the simultaneous PCR-SSP was simple, rapid, accurate and reproducible. The frequencies of HPA-3a and HPA-15a genes in Fujian Han blood donors were lower than those in other HPA systems. The frequencies of HPA-4a and -7a genes were more than 0.99999.The results showed that the frequencies of a gene of HPA-1HPA-2ON-5m6 system were all greater than 0.9500, which were 0.99380.9680.98130.9625.3. 7 strains of BLCLs were constructed, and 4 strains of HPA-15ab / 2 strain HPA-15ar-15aa / a were selected. One HPA-15b / b 1 strain was established for eight weeks. The results showed that the HPA-15 genotypes of 4 BLCLs strains were in good agreement with each other before and after establishment. The HPA-15 genotypes of BLCLs cell lines cultured in different generations were identical, and the HPA-15 genotypes of BLCLs cell lines were identical. It is proved that BLCLs is a stable and reliable reference material for HPA-15 genotyping. The establishment of BLCLs cell lines of three HPA-15 genotypes facilitates the quality control of HPA-15 genotyping. The resuscitation BLCLs can only be cultured for one week, and the results are accurate and reliable. Conclusion the synchronous PCR-SSP method of HPA genotyping can be established by reasonably designing primers, optimizing circulation parameters and reaction system. The gene frequencies of HPA(1~7 and 15) of Han blood donors in Fujian Province are similar to those of Korea, Vietnam, Taiwan and other countries and regions, and are similar to those of Europe. In Africa, America, Australia and other regions, the ethnic difference is great. It is feasible to establish the reference DNA cell line for HPA-15 genotyping.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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相關(guān)期刊論文 前1條

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本文編號：1601562