增强ESAT6-CFP10融合蛋白诱导小鼠细胞免疫应答佐剂的筛选

Screening of adjuvant enhancing cellular immune response induced by ESAT6-CFP10 fusion protein in mice

目的筛选能增强特异性抗原早期分泌抗原靶6蛋白(Early secretory antigenic target 6,ESAT6)-培养滤出液蛋白-10(Culture filtrate protein 10,CFP-10)融合蛋白(E1C0)诱导小鼠细胞免疫应答的佐剂,建立基于细胞免疫应答的小鼠模型,以评价基于体外干扰素γ释放分析(IFNγrelease assay,IGRA)结核诊断方法中特异性刺激抗原E1C0的活性。方法建立小鼠IFNγ双抗体夹心SABC-ELISA检测系统,并验证系统的线性、灵敏度、重复性和特异性。将BALB/c小鼠随机分为7组:E1C0+单磷酸类脂A(Monophosphoryl lipid A,MPL)+双十八烷基二甲基溴化铵(Dimethyl dioctadecylammonium bromide,DDA)组、E1C0+DDA组、E1C0+MPL组、E1C0+弗氏不完全佐剂(IFA)组、E1C0组、生理盐水组和MPL+DDA联合组,每组6只,经小鼠后肢内侧皮下免疫3次,间隔2周,免疫剂量为:E1C0 100μg/只,MPL 25μg/只,DDA 250μg/只,IFA 100μl/只。末次免疫4周后处死小鼠,无菌取脾,分离脾淋巴细胞,加入E1C0进行培养,MTT法检测特异性淋巴细胞增殖反应,ELISA法检测培养上清中IFNγ水平。采用筛选出的最佳佐剂与抗原组合免疫3批BALB/c小鼠,进行IFNγ诱生测定。结果检测系统的线性范围为:40～2 560 pg/ml(R>0.98);灵敏度为40 pg/ml;变异系数(CV)<15%,检测大鼠、豚鼠和兔血清IFNγ均为阴性;E1C0+MPL+DDA组、E1C0+IFA组和E1C0+DDA组小鼠特异性淋巴细胞增殖刺激指数均明显高于生理盐水组(P<0.01);E1C0+MPL+DDA组小鼠的脾淋巴细胞经E1C0体外刺激后,产生的IFNγ水平明显高于其他组(P<0.001);重复试验结果显示,E1C0+MPL+DDA组3批小鼠免疫后,脾淋巴细胞诱生的IFNγ水平差异无统计学意义(P>0.05)。结论 E1C0与MPL和DDA联合免疫所诱导的小鼠Th1型细胞免疫应答最强,成功建立了用于评价刺激抗原E1C0活性的小鼠模型。

Objective To screen the adjuvant enhancing the cellular immune response induced by early secretory antigenic target 6（ESAT6）-culture filtrate protein-10（CFP10）in mice,and establish an animal model based on cellular immune response for evaluation of activity of specific stimulating antigen E1C0 in IFNγ release assay（IGRA）for diagnosis of tuberculosis（TB）.Methods Double antibody sandwich SABC-ELISA system for mouse IFNγ was developed and verified for linearity,sensitivity,reproducibility and specificity.BALB / c mice were randomly divided into seven groups,6 for each,and immunized s.c.with E1C0 ＋ monophosphoryl lipid A（MPL）＋ dimethyl dioctadecylammonium bromide（DDA）,E1C0 ＋ DDA,E1C0 ＋ MPL,E1C0 ＋ IFA,E1C0,physiological saline and MPL ＋ DDA for 3 times,respectively,each at an interval of 2 weeks.The dosages of E1C0,MPL,DDA and IFA for immunization were 100 μg,25 μg,250 μg and 100 μl,respectively.The mice were killed 4 weeks after the last immunization,and their spleens were collected aseptically,from which splenic lymphocytes were isolated,cultured with E1C0,then determined for proliferation level by MTT method,and for IFNγ level in culture supernatant by ELISA.Three batches of BALB/c mice were immunized with the screened adjuvant combined with antigen,and determined for IFNγ induced.Results The linear range,sensitivity and CV value of developed SABC-ELISA system were 40 ~ 2 560 pg / ml（R 0.98）,40 μg / ml and less than 15% respectively,by which all the detection results of IFNγ in rat,guinea pig and rabbit sera were negative.The stimulating indexox（SIs） of specific lymphocyte proliferation in E1C0 ＋ MPL ＋ DDA,E1C0 ＋ IFA and E1C0 ＋ DDA groups were significantly higher than those in physiological saline group（P 0.01）.The IFNγ level secreted by lymphocytes in E1C0 ＋ MPL ＋ DDA group after stimulation with E1C0 in vitro was significantly higher than those in other groups（P 0.001）.No significant differences were observed in IFNγ levels induced in 3 batches of mice in E1C0 ＋ MPL ＋ DDA group（P 0.05）.Conclusion The immunization with E1C0 in a combination with MPL and DDA elicited a strong Th1 cellular immune response in mice.Mouse model for evaluation of activity of E1C0 antigen was successfully established.