汉滩病毒核蛋白T细胞表位的鉴定

发布时间：2018-08-14 11:30

【摘要】：目的：鉴定出汉滩病毒核蛋白(HTNV-NP)上的CD4~+T细胞和CD8~+T细胞表位。方法：(1)用HTNV-NP部分重叠合成肽体外刺激HFRS患者PBMC，应用ELISPOT方法筛选出可刺激HFRS患者PBMC产生IFN-γ的合成肽组，，再筛选出该组中的单个阳性15肽。(2)用EB病毒转化HFRS患者PBMC，建立EBV转化的B淋巴母细胞系(B-LCL)。用免疫磁珠分离HFRS患者PBMC中的CD4~+T细胞或CD8~+T细胞作为效应细胞，与加载过阳性15肽的自身B-LCL共同孵育，应用ELISPOT方法确定15肽可否刺激CD4~+T细胞或CD8~+T细胞产生IFN-γ，可刺激CD4~+T细胞产生IFN-γ的15肽即为CD4~+T细胞表位，可刺激CD8~+T细胞产生IFN-γ的15肽中，则可能包含有CD8~+T细胞表位。(3)应用生物信息学对可刺激CD8~+T细胞产生IFN-γ的15肽序列中包含的9肽表位进行预测，用蛋白质自动合成仪合成预测9肽，并应用ELISPOT鉴定出其中可刺激CD8~+T细胞产生IFN-γ的9肽CTL表位。(4)用HLA-DR、DP和DQ的阻断抗体与PBMC预先孵育30min后，再加入15肽，共同孵育过夜，应用ELISPOT阻断试验确定CD4~+T细胞表位的HLA-DR、DP或DQ限制性。(5)以
[Abstract]:Aim: to identify CD4 ~ T cells and CD8 ~ T cell epitopes of Hantaan virus nucleoprotein (HTNV-NP). Methods: (1) PBMCs of HFRS patients were stimulated by partial overlapping synthetic peptide of HTNV-NP in vitro. The synthetic peptide groups which could stimulate PBMC production of IFN- 纬 in HFRS patients were screened by ELISPOT method. Then a single positive 15 peptide was screened. (2) B lymphoblastoid cell line (B-LCL) transformed by EBV was established by transforming EBV into PBMCs of HFRS patients. CD4T cells or CD8T cells isolated from PBMC of HFRS patients were isolated by immunomagnetic beads as effector cells and incubated with B-LCL loaded with positive 15-peptide. The ELISPOT method was used to determine whether the 15-peptide can stimulate the production of IFN- 纬 by CD4T cells or CD8T cells. The 15 peptides that can stimulate the production of IFN- 纬 by CD4T cells are CD4T cell epitopes, among the 15 peptides that stimulate CD8T cells to produce IFN- 纬. It may contain CD8T cell epitopes. (3) bioinformatics was used to predict 9 peptide epitopes contained in 15 peptide sequences that stimulate CD8T cells to produce IFN- 纬. ELISPOT was used to identify 9 peptide CTL epitopes which could stimulate CD8 ~ T cells to produce IFN- 纬. (4) 30min was preincubated with PBMC with HLA-DRDP and DQ blocking antibodies, and then 15 peptides were added to it for overnight incubation. ELISPOT blocking assay was used to determine HLA-DRN DP or DQ restriction of CD4 ~ T epitopes.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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