【摘要】 目的 用结核分枝杆菌（Mycobacterium tuberculosis）特异性抗原Rv3400联合弗氏不完全佐剂（incomplete Freund’s adjuvant,IFA）构建结核亚单位疫苗（Rv3400-IFA）,在C57BL/6小鼠体内评估其免疫效应。体外用抗原Rv3400感染巨噬细胞RAW264.7并评估其免疫应答。方法 PCR扩增基因Rv3400,构建重组质粒pET28a（+）-Rv3400,转入大肠埃希菌BL21（DE3）pLysS感受态细胞中,诱导表达并纯化Rv3400蛋白。用抗原Rv3400、阳性对照脂多糖（lipopolysaccharides,LPS）,分别刺激小鼠巨噬细胞RAW264.7,阴性对照为未刺激的巨噬细胞,流式细胞术检测细胞表面分子表达,收集细胞培养上清,ELISA检测细胞因子的分泌。另一方面纯化的Rv3400蛋白与IFA充分乳化构建亚单位疫苗免疫小鼠;C57BL/6小鼠采用数字表法随机分为3组,分别为实验组Rv3400组、阳性对照组Rv3425组、阴性对照组IFA组,每组5只,每2周1次、共免疫3次。分别采用酶联免疫斑点检测技术（enzyme-linked immunospot assay,ELISPOT）和酶联免疫吸附测定（ELISA）技术对免疫小鼠的细胞免疫和体液免疫进行评估。结果（1）成功克隆表达并纯化结核分枝杆菌特异性抗原Rv3400。（2）体外细胞实验结果显示:Rv3400抗原刺激巨噬细胞,其表面分子CD80、CD86、CD40、MHCⅡ表达量显著提高,阳性细胞比率[分别由未刺激的（34.70±2.40）%、（31.25±18.31）%、（41.80±6.01）%、（44.30±0.44）%提高至1μg/ml Rv3400抗原刺激后的（90.45±7.71）%、（90.10±4.10）%、（89.97±7.79）%、（85.13±5.12）%];能促进细胞分泌高水平的肿瘤坏死因子（tumor necrosis factor-α,TNF-α）和白细胞介素-6（interleukin-6,IL-6）,分别达（49 217.0±390.61）pg/ml、（1783.4±51.18）pg/ml。（3）免疫小鼠后,ELISPOT结果显示:实验组Rv3400组小鼠分泌INF-γ的斑点形成细胞（spotforming cells,SFC）（136.20±95.09）显著高于阴性对照组（14.00±9.62）（t=2.859,P<0.01）;ELISA结果显示:Rv3400组小鼠T细胞产生TNF-α和IFN-γ的水乎[（247.38±142.268）pg/ml、（325.45±75.19）pg/ml)]明显高于阴性对照组[（69.25±60.06）pg/ml、（2.22±1.28）pg/ml（t=2.579,P<0.05;t=8.597,P<0.01）];（4）Rv3400组小鼠脾CD4⁺CD44^high、CD4⁺CD62L^low、CD8⁺CD44^high、CD8⁺CD62L^lowT淋巴细胞百分比[（44.20±11.95）%,（43.56±11.90）%,（48.88±7.42）%,（47.04±7.72）%]与对照组IFA组[（27.02±6.56）%,（62.60±4.73）%,（30.57±6.00）%,（58.48±6.06）%]相比,差异有统计学意义（one-way ANOVA,F=18.460,F=8.108,F=32.194;F=4.080;P<0.05,P<0.01,P<0.01,P<0.05）,呈显著性增加。（5）通过间接ELISA测定免疫后的小鼠血清抗体IgG效价水平高达1:409 600,表明抗原Rv3400也能诱导有效的体液免疫应答;IgG2b/IgG1水平为1.8,说明抗原Rv3400诱导小鼠以Th1型免疫反应为主。结论 亚单位疫苗Rv3400-IFA在C57BL/6小鼠体内可产生较好的细胞免疫和体液免疫效应。结核分枝杆菌特异性抗原Rv3400有望成为新的结核病疫苗设计及诊断的候选抗原。更多还原

【Abstract】 Objective To construct a subunit vaccine（Rv3400-IFA） by combining specific antigen Rv3400 of Mycobacterium tuberculosis with incomplete Freund’s adjuvant（IFA）,to assess its immune effects in C57BL/6 mice and by infecting the macrophages RAW264.7 with antigen Rv3400.Methods The Rv3400 gene fragment was amplified by PCR,and inserted into plasmid pET28a（+）.The protein Rv3400 was expressed in E.coli BL21（DE3）pLysS and purified.The macrophages RAW264.7 were divided into 3 groups including positive control group[treated with 1 fig/ml lipopolysaccharides（LPS）],test group（treated with 1 μg/ml Rv3400）,negative control group（no treated）,the expression of cell surface markers was examined by flow cytometry,and cell culture supernatant was collected and detected the cytokines secreted by ELISA.Rv3400 protein and IFA were equally mixed and emulsified.The C57BL/6 mice were randomly divided into 3 groups with each group 5 mice including test group（treated with Rv3400-IFA）,positive control group（treated with Rv3425-IFA）,negative control group（treated with IFA）,immunized 3 times at 2 week interval.The humoral and cellular immune responses in mice were analysed by enzymelinked immunospot assay（ELISPOT）,ELISA and flow cytometry.Results（1） The Rv3400 protein was successfully expressed and purified.（2） The macrophages were stimulated with 1 fig/ml Rv3400 antigen,significantly improved the expression of surface markers CD80[from（34.70 ± 2.40）%to（90.45±7.71）%],CD86[from（31.25±18.31）%to（90.10±4.10）%],CD40[from（41.80±6.01）%to（89.97±7.79）%]and MHCⅡ[from（44.30±0.44）%to（85.13±5.12）%],and promoted pro-inflammatory cytokine production[TNF-α（49 217.0±390.61）pg/ml,IL-6（1783.4±51.18）pg/ml].（3） The spot forming cells（SFC） of IFN-γ in mice immunized with Rv3400-IFA was（136.20±95.09）,significantly higher than that in IFA group（14.00 ± 9.62）（t=2.859,P<0.01）.The TNF-a and IFN-γ levels in spleen lymphocyte culture supematants were（247.38±142.268）pg/ml and（325.45±75.19） pg/ml respectively,significantly higher than those in IFA group[（69.25±60.06） pg/ml and（2.22±1.28）pg/ml]（t=2.579,P<0.05^=8.597,P<0.01）.（4） The percentages of CD4⁺ CD44^high[（44.20±11.95）%],CD4⁺CD62L^low[（43.56 ± 11.90）%],CD8⁺CD44^high[（48.88±7.42）%]and CD8⁺CD62L^low T cells[（47.04±7.72）%]in Rv3400-IFA group were significantly higher than those in IFA group[（27.02±6.56）%,（62.60±4.73）%,（30.57±6.00）%and（58.48 ± 6.06）%],（one-way ANOVA,F= 18.460,F= 8.108,F =32.194;F=4.080;P<0.05,P<0.01,P<0.01,P<0.05）.（5） The serum IgG level in test group was as high as1:409 600.The level of IgG2b/IgGl was 1.8.Conclusion The subunit vaccine Rv3400-IFA can induce strong humoral and cellular immune responses in C57BL/6 mice.Mycobacterium tuberculosis Rv3400 protein may be a novel candidate antigen for designing the TB vaccine and diagnosing the TB.更多还原