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目的筛选和确定TTK-EG包含的H-2~d限制的HIV-1特异性T细胞表位。方法 6~8周龄雌性BALB/c小鼠0周时分别经肌肉注射、皮内注射和滴鼻途径免疫5×10~6PFU TTK-EG,间隔4个月进行加强免疫。末次免疫后2周取小鼠脾细胞,采用IFN-γELISPOT方法筛选HIV-1 Env和Gag肽库,反应阳性的肽库进行第二轮单肽筛选。用流式细胞术(胞内因子染色,intracellular cytokine staining,ICS)对筛选出的单肽进行验证(小鼠免疫途径为肌肉注射),确定H-2~d限制的HIV-1特异性T细胞表位。结果经过ELISPOT筛选和ICS验证,确定2条Env多肽(gp140-9,氨基酸位点60~77;gp140-74,氨基酸位点548~565)和3条Gag多肽(Gag-49,氨基酸位点196~210;Gag-64,氨基酸位点256~270;Gag-65,氨基酸位点260~274)包含H-2~d限制的HIV-1特异性T细胞表位,其中gp140-74(IVQQQSNLLRAIEAQQHL)是首次发现的H-2~d限制的HIV-1特异性T细胞表位。Env、Gag优势表位肽与Env、Gag全肽池刺激小鼠T细胞分泌IFN-γ、IL-2、TNF-α三种细胞因子的能力基本相同。结论 Env多肽gp140-9、gp140-74和Gag多肽Gag-49、Gag-64、Gag-65包含H-2~d限制的T细胞表位,可用于重组痘苗病毒TTK-EG的T细胞免疫研究。
Objective To screen and identify H-2 ~ d-restricted HIV-1-specific T cell epitopes contained in TTK-EG. Methods Female BALB / c mice of 6-8 weeks old were immunized with 5 × 10 ~ 6 PFU TTK-EG by intramuscular injection, intradermal injection and intranasal instillation at 4 weeks after immunization. Mice spleen cells were taken 2 weeks after the last immunization, HIV-1 Env and Gag peptide library were screened by IFN-γELISPOT method, and the positive peptide library was screened for the second round of single peptide screening. The selected single peptides were verified by flow cytometry (intracellular cytokine staining (ICS)) (mouse immunization route was intramuscular injection) to determine H-2 d-restricted HIV-1 specific T cells gauge. Results The two Env polypeptides (gp140-9, amino acid positions 60-77; gp140-74, amino acid positions 548-565) and three Gag polypeptides (Gag-49, amino acid position 196 ~ 210; Gag-64, amino acid positions 256 to 270; Gag-65, amino acid positions 260 to 274) contain HIV- 1 -specific T cell epitopes restricted by the H-2 ~ d, wherein gp140-74 (IVQQQSNLLRAIEAQQHL) Is the first H-2-d-restricted HIV-1-specific T cell epitope found. Env, Gag dominant epitope peptide and Env, Gag peptide pool stimulated mouse T cells secreting IFN-γ, IL-2, TNF-α three cytokines are basically the same. Conclusions Env polypeptides gp140-9, gp140-74 and Gag polypeptides Gag-49, Gag-64 and Gag-65 contain H-2 d-restricted T cell epitopes and can be used for T cell immunization of recombinant vaccinia virus TTK-EG .