结核分枝杆菌新型抗原Rv3400的免疫原性研究  

Immunologenicity of specific antigen Rv3400 of Mycobacterium tuberculosis

作　　者：朱琳[1] 徐颖[1] 孔聪[1] 粟海波[1] 黄琪[1] 朱胜玲[1] 王洪海[1] 

机构地区：[1]复旦大学生命科学学院,上海200438

出　　处：《中国防痨杂志》2016年第3期201-208,共8页Chinese Journal of Antituberculosis

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（incom-pleteFreund’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，转人大肠埃希菌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（1ipop01ysaccharides，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-linkedimmunospotassay，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/mlRv3400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（tumornecrosisfactor-a，TNF-a）和白细胞介素-6（interleukin-6，IL-6），分别达（49217．0±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-7的斑点形成细胞（spotfoObjective 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 [trea- ted with 1 /ag/ml lipopolysaccharides （LPS）], test group （treated with 1 /~g/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 emul- sified. 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 enzyme linked immunospot assay （ELISPOT）, ELISA and flow cytometry. Results （1） The Rv3400 protein was suc- cessfully expressed and purified. （2） The macrophages were stimulated with 1 ~g/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-a （49 217.0±390.61）pg/ml, IL-6 （1783.44-51.18）pg/ml]. （3） The spot forming cells （SFC） of IFN-y 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,

关 键 词：分枝杆菌 结核 Rv3400 亚单位疫苗 细胞免疫 体液免疫 

分 类 号：R378.911[医药卫生—病原生物学]