结核分枝杆菌Rv2941蛋白抗原表位集中区免疫原性研究  

Evaluation on the immunogenicity of the antigen epitopes concentrated areas of Rv2941 protein from Mycobacterium tuberculosis

作　　者：范雪亭 栾秀丽 赵秀芹[1] 李马超[1] 万康林[1] 卢选成[2] 李晓燕[2] 刘海灿[1] FAN Xue-ting;LUAN Xiu-li;ZHAO Xiu-qin;LI Ma-chao;WAN Kang-lin;LU Xuan-cheng;LI Xiao-yan;LIU Hai-can(State Key Laboratory for Infectious Disease Prevention and Control,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Laboratory Animal Center,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206 [2]中国疾病预防控制中心实验动物中心,北京102206

出　　处：《中国人兽共患病学报》2022年第5期394-399,共6页Chinese Journal of Zoonoses

基　　金：“十三五”传染病重大专项(No.2018ZX10731301-002)。

摘　　要：目的分析结核分枝杆菌Rv2941抗原的T细胞表位集中区的免疫原性,探究其作为结核病疫苗候选抗原的潜力。方法通过免疫表位数据库(Immune Epitope Database,IEDB)分析Rv2941抗原的T细胞表位区(命名为Rv2941p)并构建表达载体PET32a-Rv2941p,诱导表达并纯化Rv2941p。将其与免疫佐剂二甲基三十六烷基铵(DDA)和PolyI:C乳化后,皮下免疫BALB/c小鼠3次,每次免疫间隔10 d,末次免疫1周后处死小鼠,进行免疫效果评价。采用ELISA法检测免疫后小鼠血清中IgG、IgG1和IgG2a抗体滴度以及免疫后小鼠脾脏淋巴细胞分泌IL-4、IL-2、IL-6和IFN-γ的水平。同时,利用流式细胞技术检测淋巴细胞内CD4^(+)T、CD8^(+)T细胞增殖情况以及胞内细胞因子(IFN-γ、TNF-α和IL-4)表达水平。结果Rv2941p可溶性表达,并获得高纯度的蛋白。Rv2941p诱导产生了高水平的特异性抗体IgG,与对照组相比,差异有统计学意义(P<0.001)。另外,Rv2941p提高了IgG2a/IgG1的比值。细胞因子检测结果显示,Rv2941p提高了IFN-γ和IL-6的分泌,与Ag85B组比较,差异有统计学意义(P<0.001)。同时,Rv2941p可以促进CD4^(+)和CD8^(+)T细胞的增殖分化,以及提高胞内IFN-γ和TNF-α的表达。结论Rv2941p可以刺激小鼠产生较高的体液和细胞免疫,尤其Th-1类细胞免疫,可以作为结核病疫苗候选抗原,为结核病新型疫苗的开发奠定了基础。To analyze the immunogenicity of the antigen epitopes concentrated areas of Rv2941 protein from Mycobacterium tuberculosis,and investigate its potential candidate antigen for tuberculosis vaccine.T cell epitopes of Rv2941 antigen was analyzed by Immune Epitope Database(IEDB).The antigen epitopes concentrated areas(649-1017 bp,named Rv2941p)of Rv2941 gene was inserted into pET32a vector,then expressed in Escherichia coli(E.coli)(DE3)BL21 cells.Prokaryotic expression showed that this protein was mainly in the form of soluble form.BALB/c mice were immunized subcutaneously in triplicate over a 10-day interval.One week after the last imm unization,the samples were collected to assess the immunogenicity.Rv2941p elicited significantly higher antigen-specific immunoglobulin G(IgG)than control groups(P<0.001),indicating that Rv2941p enhanced antibody response.To evaluate the cell mediated immune response,the cytokines(e.g.,IL-4,IL-2,IL-6 and IFN-γ)were detected by ELISA.These results showed that Rv2941p significantly improved the level of IFN-γand IL-6 compared with Ag85B(P<0.001).Meanwhile,the results of IgG subclass shown that Rv2941p could improve the ratio of IgG2a/IgG1.And the percentage of splenic CD4^(+)T and CD8^(+)T cells were measured by flow cytometry.In addition,the intracellular cytokines(e.g.,IFN-γ,TNF-αand IL-4)were detected by flow cytometry.We found that Rv2941p enhanced intracellular expression of IFN-γand TNF-αcytokines,and promoted CD4^(+)T cell and CD8^(+)T cell proliferation.Further this study provided evidence that the T cell epitopes of Rv2941 promoted stronger cell-mediated immune response,and skewed to Th-1 type cellular immune response.In conclusion,the T cell epitope of Rv2941 could promote humoral and cell-mediated immune responses,which supports it as a promising antigen for TB vaccines.

关 键 词：结核分枝杆菌 Rv2941 T细胞表位 结核病疫苗 

分 类 号：R378.91[医药卫生—病原生物学]