结核分枝杆菌PE31抗原的免疫原性及保护效果研究

摘要/Abstract

摘要： 目的选择结核分枝杆菌中受PhoPR和Lsr2调控的PE-PPE家族蛋白PE31作为研究对象,探索其免疫原性和抗结核免疫保护效果,为新型抗结核疫苗的研发提供候选抗原。方法根据PE31的氨基酸序列预测其CD4+、CD8+T细胞表位。在大肠杆菌和哺乳动物细胞系中分别表达其编码基因,通过Western-blotting实验验证目的蛋白表达。联合弗氏佐剂免疫C57BL/6小鼠,通过比较脾细胞抗原特异性IFN-γ、TNF-α和IL-2的分泌水平,以及小鼠血清IgG水平,评价抗原免疫原性。以DNA疫苗形式免疫BALB/c小鼠后,尾静脉感染结核分枝杆菌标准毒株H37Rv,通过比较4周后小鼠体内肺、脾载菌量评价其保护效果。结果 PE31与Ag85A携带有相同数量的T细胞表位。并且能够诱导出强于Ag85A的IFN-γ、TNF-α 细胞因子分泌和相同的血清IgG反应水平。PE31免疫组小鼠肺、脾结核杆菌载量比未免疫组下降0.3~0.4log₁₀。表现出更强的细菌清除能力和免疫保护能力。结论 PE31具有较强的免疫原性和免疫保护能力,本研究成果将为抗结核疫苗的开发提供新的候选抗原及抗原表位。

关键词: 结核病, 亚单位疫苗, PE31, 潜伏感染

Abstract: Objective The purpose of this study is to examine the immunogenicity and protective efficacy of PE31, which is a PE-PPE family protein that is under the control of the PhoPR two-component system and Lsr2 global regulator, against Mycobacterium tuberculosis(MTB); and to evaluate its potential in subunit vaccine development. Methods The sequence of PE31 was analyzed for potential CD4⁺ and CD8⁺T cell epitopes. PE31 and Ag85A were cloned into pET-28a and recombinant proteins were expressed in and purified from E. coli. C57BL/6 mice were immunized with purified antigens formulated in adjuvant and the production of antigen-specific Th1 cytokines and antibody were determined. PE31 and Ag85A were also cloned into pcDNA3.1(+) for mammalian cell expression. The resulting DNA vaccines were used to immunize BALB/c mice subcutaneously. The mice were then challenged intravenously by MTB H37Rv. Bacterial burdens in the lungs and spleen were determined 4 weeks post-infection. Results There are similar numbers of predicted CD4⁺ and CD8⁺T cell epitopes in PE31 and Ag85A. Immunization experiments showed that PE31 induced higher level of IFN-γ and TNF-α (~600 pg/ml) than Ag85A and similar levels of antibody response. Mice immunized with the DNA vaccine expressing PE31 had 0.3-0.4log₁₀ fewer MTB counts in the lungs and spleen than those immunized with the empty vector. Conclusions It is demonstrated that PE31 is highly immunogenic and protective, and is an attractive candidate for the development of novel subunit vaccines.

Key words: Tuberculosis, Subunit vaccine, PE31, Latent infection

王玉臣, 陈福增, 黄滢睿, 张鹭, 刘军. 结核分枝杆菌PE31抗原的免疫原性及保护效果研究[J]. 新发传染病电子杂志, 2017, 2(3): 155-159.

WANG Yu-chen, CHEN Fu-zeng, HUANG Ying-rui, ZHANG Lu, LIU Jun. Immunogenicity and protective efficacy of mycobacterium tuberculosis antigen PE31[J]. Electronic Journal of Emerging Infectious Diseases, 2017, 2(3): 155-159.

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