复制型重组痘苗病毒TTK-EG特异性T细胞表位研究  

Specific T-cell epitope mapping of replication-competent recombinant vaccinia virus TTK-EG

作　　者：于海[1] 王书晖[1] 刘颖[1] 王硕[1] 邵一鸣 YU Hai WANG Shuhui LIU Ying WANG Shuo SHAO Yiming(National Center for AIDS/STD Control and Prevention, State Key Laboratory for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beifing 102206,China)

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,传染病预防控制国家重点实验室,北京102206

出　　处：《中国热带医学》2017年第5期435-439,469,共6页China Tropical Medicine

基　　金：“十二五”科技重大专项“预防性艾滋病疫苗”(No.2012ZX10001008);传染病预防控制国家重点实验室基金(No.2012SKLID103)

摘　　要：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 the H-2^d restricted HIV-1 specific T-cell epitopes in TTK-EG. Methods6-8 week-old female BALB/c mice were primed with 5×10^6 PFU of TTK-EG at month 0 intramuscularly,intracutaneously andintranasally,respectively,and then boosted at month 4.2 weeks after the final inoculation,the spleen cells were isolated andtested by IFN-γ ELISPOT using the Env and Gag peptide pools.The positive pools were further screened to identify thedominant peptides.ICS was used to confirm that the screened peptides were H-2^d restricted HIV-1 specific T-cell epitopes ornot（female BALB/c mice were immunized by intramuscular injection）. Results Two Env-specific（gp140-9, aa 60-77;gp140-74,aa 548-565） and three Gag-specific（ Gag-49, aa 196-210; Gag-64, aa 256-270; Gag-65, aa 260-274） peptideswere identified containing HIV-1 specific T-cell epitopes,and gp140-74（IVQQQSNLLRAIEAQQHL） was first discoveredcontaining H-2^d restricted T-cell epitopes.The ability to stimulate HIV specific CD8^＋T cells secreting IFN-γ, IL-2 and TNF-α cytokines between the immunodominant epitope peptides and all-peptide pools was comparative. Conclusion The Envpeptides gp140-9, gp140-74 and Gag peptides Gag-49, Gag-64, Gag-65 contained H-2^d restricted T-cell epitopes,and couldbe used to study the T cell responses in BALB/c mice.

关 键 词：HIV-1 重组痘苗病毒 T细胞表位 

分 类 号：R512.91[医药卫生—内科学]