本文選題：HLA + 超型　； 參考：《第三軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：研究背景:由乙型肝炎病毒(HBV)引起的慢性乙型肝炎是我國常見的慢性傳染病之一,嚴(yán)重危害人民健康。以往研究表明,抗原特異性的CD8+細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocyte,CTL)對體內(nèi)HBV的清除起關(guān)鍵作用。在乙肝患者體內(nèi),來源于HBV相關(guān)抗原的、MHC-I類分子限制性表位肽被認(rèn)為是特異性CD8+CTL識(shí)別病毒感染細(xì)胞的靶標(biāo)。雖然慢性HBV感染患者外周血中存在抗原肽特異性CTL,但這種病毒特異性CD8+CTL的頻率及功能減低,是HBV得以持續(xù)存在的一個(gè)重要原因。因此,通過主動(dòng)免疫方式增強(qiáng)HBV相關(guān)抗原肽特異性CTL反應(yīng),是治療慢性HBV感染的理想策略。既往對于病毒抗原表位肽的研究多針對單一主要組織相容性復(fù)合體(Major histocompatibility complex, MHC)限制性,缺乏覆蓋更多人白細(xì)胞抗原(human leucocyte antigen ,HLA)背景人群的治療性表位肽疫苗。HLA分子的高度多態(tài)性與T細(xì)胞識(shí)別的MHC限制性是研制廣譜高效表位肽疫苗的最大障礙。HLA超型是指具有相同的肽結(jié)合特異性的不同HLA分子的總合。這一概念的提出,為基于HBV相關(guān)抗原超型表位肽疫苗的設(shè)計(jì)提供了新的思路。 研究目的:本研究初步分析了慢性HBV感染與HLA-A超型的相關(guān)性,預(yù)測并初步鑒定了HLA-A3超型限制性HBV相關(guān)抗原特異性CTL表位肽,為基于超型表位的慢性HBV感染的治療提供實(shí)驗(yàn)基礎(chǔ)及新的候選靶標(biāo)。 研究方法:分別從西南醫(yī)院肝炎門診和獻(xiàn)血中心收集慢性HBV感染者及健康志愿者外周血,通過聚合酶鏈反應(yīng)/序列特異性引物技術(shù)(PCR-SSP)對其HLA型別進(jìn)行檢測,分析慢性HBV感染與HLA-A超型之間的關(guān)系。基于SYFPEITHI、BIMAS、SVMHV、IEDB、EPIJEN、NETMHC 6個(gè)表位預(yù)測網(wǎng)站,分別預(yù)測出HLA-A3超型各等位基因(HLA-A*0301、HLA-A*1101、HLA-A*3101、HLA-A*3301、HLA-A*6801)限制性表位,并對預(yù)測結(jié)果進(jìn)行整合,預(yù)測出與HLA-A3超型各等位基因具有高親和力的乙肝表面抗原(HBsAg)和乙肝核心抗原(HBcAg)候選表位肽。鑒于HLA超型分子具有結(jié)合相同或相似表位肽的特點(diǎn),并且在HLA-A3超型中,HLA-A*1101分子表型頻率最高,因此,我們選擇首先鑒定候選超型表位肽HLA-A*1101限制性。在體外細(xì)胞功能試驗(yàn)中,通過Elispot Assay檢測候選表位肽誘導(dǎo)外周血單核細(xì)胞(PBMC)IFN-γ的分泌;胞內(nèi)因子染色流式細(xì)胞術(shù)分析表位肽特異性CD8+CTL IFN-γ及GranB的分泌;CFSE標(biāo)記檢測表位肽誘導(dǎo)的特異性CD8+CTL的增殖;Pentamer染色流式細(xì)胞術(shù)分析法檢測HLA-A*1101健康志愿者PBMC經(jīng)肽誘導(dǎo)后所產(chǎn)生的特異性CD8+CTL頻率,以及慢性HBV感染患者外周血表位肽特異性CD8+CTL的頻率。最后通過Elispot assay初步鑒定候選表位肽對其他HLA-A3超型等位基因的健康人PBMC所產(chǎn)生的誘導(dǎo)效應(yīng)。 研究結(jié)果:在我們收集的44例來自西南醫(yī)院獻(xiàn)血中心的健康志愿者中,HLA-A3超型為主要HLA-I類分子超型基因,其中以HLA-A*1101頻率最高,在49例慢性HBV感染者中,HLA-A2超型為最高頻超型等位基因。結(jié)合多個(gè)在線表位預(yù)測網(wǎng)站,采用整合法預(yù)測的超型表位兼顧了與HLA-A3超型全部等位基因具有高親和力的表位(P5, P6, P7, P8, P9)。根據(jù)應(yīng)答指數(shù)以及胞內(nèi)因子染色結(jié)果證實(shí)五條候選表位肽均能在體外誘導(dǎo)HLA-A*1101健康人外周血表位肽特異性CD8+CTL增殖并分泌IFN-γ和GranB。在HLA-A*1101陽性的慢性HBV感染患者中,P7肽誘導(dǎo)產(chǎn)生的特異性CD8+CTL頻率明顯高于已知的陽性表位P6肽。五條候選超型表位肽均可以誘導(dǎo)帶有HLA-A3超型等位基因個(gè)體的PBMC分泌IFN-γ,但是對同屬于HLA-A3超型的不同HLA等位基因,其限制性CTL分泌IFN-γ的能力存在差異。 研究結(jié)論:結(jié)合本研究中各部分的實(shí)驗(yàn)結(jié)果,HLA-A3超型在人群中分布頻率最高,同時(shí),HLA-A*1101是HLA-A3超型中最高頻等位基因。利用整合法預(yù)測的5條HBV相關(guān)抗原候選表位,經(jīng)初步鑒定為HLA-A3超型限制性CTL表位。而且,從表位肽特異性CTL的頻率與功能水平上分析,較目前已知的HLA-A*1101限制性陽性表位肽(P6),P7肽是一個(gè)明確的免疫優(yōu)勢性的HLA-A*1101限制性表位肽。
[Abstract]:Background: chronic hepatitis B caused by hepatitis B virus (HBV) is one of the common chronic infectious diseases in China, which seriously endangers the people's health. Previous studies have shown that the antigen specific CD8+ cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) plays a key role in the clearance of HBV in the body. In hepatitis B patients, it comes from HBV phase. Antigenic, MHC-I class molecular restrictive epitopes are considered to be specific CD8+CTL targets for identifying virus infected cells. Although antigenic peptide specific CTL exists in peripheral blood of patients with chronic HBV infection, the frequency and function of this virus specific CD8+CTL is an important reason for the persistence of HBV. Pestilence enhanced HBV related antigen peptide specific CTL response, which is an ideal strategy for the treatment of chronic HBV infection. Previous studies of viral antigen epitopes are mostly limited to a single major histocompatibility complex (Major histocompatibility complex, MHC), and lack of more human leukocyte antigen (human leucocyte antigen, HLA) back. The high polymorphism of the therapeutic epitope peptide vaccine.HLA molecule and the MHC restriction of T cell recognition are the biggest obstacle to the development of the broad spectrum epitope peptide vaccine.HLA super type is the combination of the different HLA molecules with the same peptide binding specificity. This concept is designed for the design of the super epitope peptide vaccine based on the HBV related antigen. A new way of thinking is provided.
Objective: This study preliminarily analyzed the correlation between chronic HBV infection and HLA-A super type, and predicted and preliminarily identified the HLA-A3 super restrictive HBV related antigen specific CTL epitope, providing experimental basis for the treatment of chronic HBV infection based on super epitopes and a new candidate target.
Methods: the peripheral blood of chronic HBV infected persons and healthy volunteers were collected from the Southwest Hospital hepatitis clinic and blood donation center respectively. The HLA types were detected by polymerase chain reaction / sequence specific primer technique (PCR-SSP), and the relationship between the chronic HBV infection and the HLA-A type was analyzed. Based on SYFPEITHI, BIMAS, SVMHV, IEDB, EPIJEN, NETMHC. 6 epitope prediction sites were used to predict the restriction epitopes of HLA-A3 super type alleles (HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*3301, HLA-A*6801), and the prediction results were integrated to predict the high affinity hepatitis B surface antigen (HBsAg) and the hepatitis B core antigen (HBcAg) candidate epitope with the HLA-A3 super type alleles. In view of the characteristics of HLA super type molecules having the same or similar epitopes and the highest phenotypic frequency of HLA-A*1101 molecules in the HLA-A3 super type, we chose to identify the HLA-A*1101 restriction of the candidate super epitopes first. In vitro cell function test, the candidate epitope was detected by Elispot Assay to induce peripheral blood mononuclear cells (P). BMC) secretion of IFN- gamma; intracellular factor dyed flow cytometry analysis of the secretion of epitope specific CD8+CTL IFN- gamma and GranB; CFSE markers to detect the proliferation of specific CD8+CTL induced by epitope peptide; Pentamer staining flow cytometry analysis of the specific CD8+CTL frequency of PBMC in HLA-A*1101 healthy volunteers after peptide induction. The frequency of peripheral blood epitoped peptide specific CD8+CTL in patients with chronic HBV infection. Finally, the inducement effect of candidate epitopes on the healthy human PBMC of other HLA-A3 super type alleles was preliminarily identified by Elispot assay.
Results: in 44 healthy volunteers from the blood donation center of Southwest Hospital, the HLA-A3 super type was the main HLA-I molecular super type gene, of which the frequency of HLA-A*1101 was the highest. Among the 49 cases of chronic HBV infection, the HLA-A2 super type was the most high-frequency super type allele. The super epitopes have high affinity to all alleles of HLA-A3 super type (P5, P6, P7, P8, P9). According to the response index and intracellular factor staining results, it is confirmed that all five candidate epitopes can induce the proliferation of the peripheral blood epitoped peptide specific CD8+CTL in HLA-A*1101 healthy human and secrete IFN- gamma and GranB. in HLA-A*1101 in vitro. In the patients with positive chronic HBV infection, the specific CD8+CTL frequency induced by P7 peptide is significantly higher than the known positive epitopes P6 peptide. Five candidate super epitopes can induce PBMC secreting IFN- gamma with HLA-A3 super type allele, but the restrictive CTL secretes IFN- gamma for the same HLA alleles belonging to HLA-A3 super type. There is a difference in ability.
Conclusion: combined with the experimental results of various parts of this study, the HLA-A3 super type is the highest frequency in the population, while HLA-A*1101 is the most high frequency allele in the HLA-A3 super type. The 5 HBV associated antigen candidate epitopes predicted by the integration method are preliminarily identified as HLA-A3 super limited CTL epitopes. Moreover, the frequency of the epitopes specific CTL Compared with the current known HLA-A*1101 restrictive positive epitopes (P6), the P7 peptide is a definite immune dominant HLA-A*1101 restrictive epitope.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【共引文獻(xiàn)】

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本文編號(hào)：2095350