汉族人部分人类血小板抗原基因分型及参考DNA细胞系的建立

发布时间：2018-03-12 12:28

本文选题：人类血小板抗原　切入点：同种免疫　出处：《福建医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的建立人类血小板抗原HPA-1～7和-15的基因分型方法同步PCR-SSP,并以该法调查分析福建省汉族献血者HPA基因频率;建立HPA-15基因分型参考DNA细胞系,即B淋巴母细胞系(BLCLs)。方法(1).检索HPA基因序列,用Primer Premier 5.0设计HPA引物和内参引物(HGHG),每对引物进行BLAST。对HPA各系统和HGHG内参作温度梯度,探索最佳退火温度,用Touch Down调节各HPA系统和HGHG内参的反应体系。(2).经过优化的反应体系对HPA基因型已知的参考DNA标本进行分型,验证方法的准确性,建立同步PCR-SSP。(3).配制引物混合物并分装。用同步PCR-SSP调查160名福建省汉族献血者的HPA基因频率,同时验证该引物混合物的重复性。(4).饥饿培养B95-8细胞至二周左右,收集Epstein-Barr病毒。(5).用PCR-SSP筛选三种HPA-15基因型血标本。EB病毒体外转化血标本B淋巴细胞,建立永生性B淋巴母细胞系(BLCLs)。(6).提取BLCLs基因组DNA,以PCR-SSP和巢式PCR鉴定其HPA-15基因型。鉴定培养3代、10代和15代BLCLs的HPA-15基因型,验证其基因型的稳定性。结果(1).建立的同步PCR-SSP操作简便、快速,准确性和重复性均为100%。(2).福建汉族献血者HPA-3a和HPA-15a基因频率较其它HPA系统a基因低,两者分别为0.5406和0.5531,两系统的杂合子百分率分别为53.13%和45.63%;HPA-4a和-7a基因频率大于0.9999(检测结果为1.0000),HPA-1、-2、-5、-6系统a基因频率均大于0.9500,分别为:0.9938、0.9688、0.9813、0.9625。(3).共建成7株BLCLs,选择4株冻存(HPA-15a/b 2株、HPA-15a/a 1株、HPA-15b/b 1株),BLCLs平均建系时间为八周;实验证明4株BLCLs建系前后HPA-15基因型完全吻合。(4).培养不同代次(3代、10代、15代)的BLCLs细胞系HPA-15基因型完全一致,从而证实BLCLs作为HPA-15基因分型的参考物质稳定可靠。(5).三种HPA-15基因型BLCLs细胞系的建立方便了HPA-15基因分型时的质量控制,复苏后的BLCLs仅需培养一周即可提取DNA,结果准确可靠。结论合理设计引物,优化循环参数和反应体系可以建立HPA基因分型的同步PCR-SSP方法;福建汉族献血者HPA(1～7和15)的基因频率与韩国、越南、台湾等国家和地区的种族相似,与欧洲、非洲、美洲和澳洲等地区的种族差异较大;建立HPA-15基因分型的参考DNA细胞系可行。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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