MPT64 DNA疫苗对鼠结核分枝杆菌感染的保护作用论文.docVIP

　　MPT64 DNA疫苗对鼠结核分枝杆菌感染的保护作用论文 孙平，骆旭东，朱道银，唐国建 【关键词】 结核分枝杆菌 【Abstract】 AIM: To explore the protective efficacy of the DNA vaccine encoding Mycobacterium tuberculosis MPT64 in mice infected ice uscularly immunized ice munization for DNA vaccine groups and 100 d after for BCG vaccinated group. The mice in the vaccinated groups and control groups oved respectively and the number of CFU in organs and histopathologic changes ined. The antibody level, IFNγ, IL4 and the survival time in all of the mice phocytes and the spleen lymphocyte proliferation from BCG group and pcDNA/MPT64 group ber of bacterial colonies in the lungs and spleens significantly decreased at 6th onary histopathological changes matory infiltration and lung tissue necrosis, and in BCG group by granulomas and numerous macrophages, lymphocytes and a fematory infiltration and a feacrophages. The results in spleen ilar to those in lungs. The survival time of BCG vaccinated mice after challenge e of pcDNA/MPT64 group prove the protective efficacy of immunized mice against M.tuberculosis challenge. 【Key tuberculosis; MPT64; DNA vaccine 【摘要】 目的： 研究MPT64 DNA疫苗对鼠结核杆菌感染的免疫保护效果. 方法： C57BL/6小鼠36只，随机分为4 组. A组（PBS）、B组（pcDNA3.1）、C组（BCG）、D组（pcDNA/MPT64）；分别于胫前肌注射质粒DNA免疫，70 μg/次， 1次/2 L（相当于0.1 mg），含活菌1×106 CFU. 将经过小鼠体内毒力复苏的H37Rv转种于改良罗氏培养基上， 37℃保湿培养2 L的菌悬液,.freelL菌悬液含M.H37Rv 1×106 CFU活菌1 mg. 攻击时间为BCG接种后100 d和末次质粒DNA免疫后5 L菌悬液，实施攻击；攻击后6 munol, 1992;36:307). 1.2.1肺、脾荷菌量及病理学检测无菌手术取左全肺，称取一定量的左肺组织加入经过灭菌的5 mL组织匀浆器中，加2 mL灭菌生理盐水，研磨成匀浆；用40 mL/L硫酸2 mL中和,摇匀后静置15 min,以此原液作10倍梯度稀释；选取3个稀释度的悬液100 μL，分别涂布于3个经过37℃复温的改良罗氏培养基上（含BCG抑制剂2thiophenecarboxylic acid hydrazide 2 mg/L），37℃保湿培养4 g/L）包被酶标板，4℃过夜. 1 g/L牛血清白蛋白封闭30 min，洗涤后加入不同稀释度的待检血清，二抗为HRP酶标羊抗鼠IgG，DAB显色，波长490 nm测吸光度（A）值. 以正常鼠血清作阴性对照，A值0.05以上、A实验组/A对照组≥2.1为阳性. ②特异性淋巴细胞增殖实验. 分别分离各组小鼠的脾淋巴细胞，用含10 g/L FCS的RPMI 1640调整细胞密度为2×107/L，活细胞数为90%以上，200 μL/孔加入96孔细胞培养板中，实验组每孔加入5 μL PPD，对照孔不加PPD，调零孔不加淋巴细胞；37℃ 50 mL/L CO2孵箱培养68 h后，每孔加入20 μL MTT（5 g/L溶于PBS，pH 7.2），继续培养4 h后终止培养，1000 r/min离心10 min，吸