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目的建立H7N9禽流感患者恢复期T细胞受体库,寻找H7N9特异性T细胞受体变化。方法收集2013年3月至5月确诊的8例人感染禽流感H7N9患者恢复期血液以及10例健康人血液,分离基因组DNA,采用Ⅲumina He Seq2000测序平台对T细胞受体库β链(TRB)抗原互补决定区3进行高通量测序,分析TRB库的多样性等特征。结果与正常组比较,H7N9患者某些TRBV基因使用频率如TRBV30、TRBV27存在显著差异。使用主成分分析降维后,发现恢复期患者TRBV-TRBJ基因配对模式与正常人有显著差别,可用于区分H7N9患者和正常人群。患者—患者、正常人—正常人之间的共享序列数量和频率明显大于患者-正常人,且患者间TRB库具有较高相似度。多样性指数在患者组明显低于正常人群,显示在感染恢复期其免疫功能仍处于相对低下状态。同时H7N9患者中超高表达克隆明显增多,且序列间存在较高相似性。结论 H7N9恢复期患者T细胞受体库有H7N9特征性变化,这些特征将为疾病的诊断和治疗性T细胞的研究提供重要信息。
Objective To establish a T cell receptor library during convalescent phase of H7N9 bird flu in search of H7N9-specific T cell receptor. Methods Genomic DNA was isolated from 8 convalescent blood of 8 H7N9 patients infected with H5N1 and 10 healthy volunteers from March 2013 to May 2013. Genomic DNA was isolated from the T cell receptor beta chain (TRB) by using Ⅲumina He Seq 2000 sequencing platform. Antigen Complementarity Determining Region 3 High-throughput sequencing and analysis of the diversity of TRB libraries. Results Compared with the normal group, the frequencies of some TRBV genes in H7N9 patients such as TRBV30 and TRBV27 were significantly different. After using principal component analysis to reduce dimension, found that patients with convalescent TRBV-TRBJ gene pairing pattern and normal people have significant differences, can be used to distinguish between H7N9 patients and normal population. The number and frequency of shared sequences between patient-patient, normal-normal individuals were significantly greater than those of patients-normal individuals, and the TRB library among patients had higher similarity. The diversity index was significantly lower in the patient group than in the normal population, indicating that the immune function remained relatively low during the recovery period. At the same time, H7N9 patients significantly increased the number of highly expressed clones, and there is a high similarity between the sequences. Conclusion The H7N9 T cell receptor repertoire in patients with convalescent H7N9 has the characteristic changes of H7N9. These characteristics will provide important information for the diagnosis of disease and the research of therapeutic T cells.