结核分枝杆菌抗原Rv0446c人T细胞抗原表位鉴定及其免疫特征的分析

Identification and immunity characterization of human T cell epitope in the Mycobacterium tuberculosis antigen Rv0446c

目的研究结核分枝杆菌Rv0446c的抗原表位及其免疫原性,为结核病的免疫诊断技术及疫苗研发提供候选抗原及表位。方法利用生物信息学软件TE-predict和IEDB的T细胞表位预测软件对结核分枝杆菌抗原Rv0446c进行T细胞表位预测,用ELISPOT实验检测表位在2019年1月到2020年12月期间来自佛山市第四人民医院和福州肺科医院的结核病患者(50例)、肺部疾病患者(39例)及健康志愿者(55例)中的免疫反应性。将60只6周龄BALB/c小鼠随机分成10组,每组6只,分别采用PBS、血蓝蛋白(KLH)、高剂量结核分枝杆菌抗原Ag85B(50μg/只)、低剂量Ag85B(20μg/只)、高计量P120、低剂量P120、高计量P121、低剂量P121、高剂量P123、低剂量P123(P120、P121、P123高剂量为100μg/只、低剂量为50μg/只)多肽对其皮下免疫,每只小鼠免疫3次,每次间隔2周,末次免疫后4周,应用ELISA方法检测各组小鼠脾细胞产生的IFN-γ、IL-2、IL-4、IL-10细胞因子水平。结果预测的7条表位多肽中,ELISPOT试验筛选出4条阳性T细胞表位多肽P120、P121、P122、P123,在结核病患者中检测灵敏度和特异度分别为30.0%、18.0%、6.0%、22.0%和96.8%、98.9%、100%、96.8%。与对照PBS组和KLH组比较,P120多肽能刺激小鼠产生较高水平的IFN-γ、IL-4、IL-10,P121多肽刺激小鼠产生较高水平的IFN-γ和IL-4(均P<0.05),P123多肽刺激小鼠产生较高水平的IFN-γ(均P<0.05)。P120、P121、P123多肽刺激小鼠产生的IL-2的水平高于PBS组低于Ag85B-L组和Ag85B-H组(均P<0.05),但与KLH组差异无统计学意义(均P>0.05)。结论Rv0446c蛋白及其T细胞表位具有较好的免疫原性及免疫反应性,能刺激机体产生强烈的细胞免疫应答,可用于结核病的临床诊断技术研究和新型结核亚单位疫苗的构建。

Objective To investigate the epitope and immunogenicity of the Mycobacterium tuberculosis antigen Rv0446c,and to propose candidate antigens and epitopes for immunodiagnosis and vaccine development of tuberculosis.Methods The bioinformatics softwares TE predict and IEDB MHC ClassⅡBinding Prediction were used to predict the T cell epitopes of Mycobacterium tuberculosis antigen Rv0446c.ELISPOT assay was used to examine the immunogenicity of these epitopes in subjects registered to Foshan Fourth People’s Hospital and Fuzhou Chest Hospital between January 2019 and December 2020,including 50 with tuberculosis,39 with pulmonary diseases,and 55 healthy volunteers.A total of 60 six-week-old BALB/c mice were randomly divided into 10 groups(n=6 each)to receive subcutaneous immunization with PBS,keyhole limpet hemocyanin(KLH),high-dose Mycobacterium tuberculosis antigen Ag85B(50μg/mouse),low-dose Ag85B(20μg/mouse),high dose P120,low dose P120,high dose P121,low dose P121,high dose P123,and low dose P123 peptides(100μg/mouse as high dose and 50μg/mouse as low dose of P120,P121 and P123)three times weekly in two weeks apart.At four weeks after the last immunization,ELISA was used to measure the levels of spleen-derived cytokines IFN-γ,IL-2,IL-4 and IL-10 in each group of mice.Results Of the seven epitope peptides predicted,ELISPOT assay identified four positive T cell epitope peptides:P120,P121,P122,and P123.The sensitivity and specificity of detection using these peptides in tuberculotic patients were 30.0%,18.0%,6.0%,22.0%and 96.8%,98.9%,100%,96.8%.Compared with the PBS control group and KLH group,P120 led to higher levels of IFN-γ,IL-4,IL-10,P121 led to higher levels of IFN-γand IL-4(all P<0.05),and P123 led to higher level of IFN-γ(all P<0.05)generated in mice.The level of IL-2 stimulated by P120,P121,and P123 in mice were higher than those by PBS group and lower than those by low-dose or high-dose Ag85B(all P<0.05),but did not differ from those by KLH(all P>0.05).Conclusion Rv0446c protein and its T cell epitope exhibit high immunogenicity and immunoreactivity that may stimulate an intense cellular immune response in the host,and therefore can be used for clinical diagnosis of tuberculosis and development of novel tuberculosis subunit vaccines.