应用ELISPOT方法筛选确定HIV-1 B／C亚型疫苗六种抗原的H-2^d限制的T细胞表位

Peptide Mapping of H-2^d Restricted T-cell Epitope against Six Antigens of HIV- 1 Subtype B/C by ELISPOT Assay

为了筛选和确定用于检测表达HIV-1 B'/C亚型病毒6种抗原(gp160、gag、polr、evt、at和nef)的艾滋病疫苗免疫小鼠后H-2d限制的特异性T细胞表位,本研究使用表达上述6种抗原的复制型DNA疫苗和非复制型重组痘苗病毒疫苗联合免疫BALB/c小鼠,通过矩阵设计将HIV-1 B(C)亚型6种相应抗原全序列肽库分别混合成肽池,使用肽池对免疫小鼠进行IFN-γELISPOT检测,根据检测结果确定肽库中特异反应的优势表位肽。结果显示:筛选到七条针对Gag的特异表位肽,其中有5条与文献报道相同,另2条为新表位肽;筛选到3条针对Pol蛋白特异表位肽,其中一条为新表位肽;筛选到2条针对gp160特异表位肽,其中一条为新表位肽;在Nef肽库中筛选到一条新的表位肽;从Tat肽库中筛选到3条表位肽,这三条肽在肽库中是连续的序列,都包含(或部分包含)网上公布的表位序列;在Rev肽库中没有筛选到能够产生阳性反应的特异性表位肽。本研究使用IFN-γELISPOT方法筛选和确定了可用于检测表达HIV-1 B'/C亚型病毒6种抗原(gp160、gag、pol、revt、at和nef)的艾滋病疫苗免疫小鼠后H-2d限制的特异性T细胞表位。

The purpose is to screen and identify the specific H-2^d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c （H-2^d） mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B＇/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c （H-2d） mice in a prime-boost regiment. The six peptide libraries of HIV B/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-γ/ production from splenocytes of immunized mice by an enzyme-linked immunospot （IFN-γ ELISPOT） assay. The ELISPOT data indicated that two of seven Gagspecific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol -specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes （H-2a restricted） were identified by IFN-y ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B/C.