目的：评价新型结核DNA疫苗p846诱导细胞免疫应答和抵抗结核杆菌卡介苗（BCG）攻击的能力。方法：通过基因克隆技术构建结核三抗原Rv3615c、Mtb10．4和Rv2660c融合的真核表达质粒p846。选择6～8周龄 BALB／c 小鼠，随机分成4组：p846疫苗组、pcDNA3．1组、PBS 组及 BCG 组，以肌肉注射的方式分别于0、2、4、6周进行免疫，BCG 组在0周皮下免疫1次。末次免疫2周后，分别用 BrdU、ELISPOT 和FCM法检测细胞免疫效应，4周后用结核菌BCG滴鼻方式进行攻击，6周后取肺组织进行病理切片评估。结果：与PBS组和空载体pcDNA3．1组相比，p846疫苗可有效促进结核特异性淋巴细胞的增殖并显著增加脾脏IFN－γ＋T 细胞的数量，且肺组织炎症反应较较轻，其保护效果与 BCG 相当。结论：新型结核 DNA 疫苗p846免疫小鼠能引起强烈的细胞免疫应答，有效抵抗结核杆菌BCG的感染。%Objective To construct a novel M.tb DNA vaccine (p846) co-expressing mycobacterial triple antigens including Rv3615c, Mtb10.4 and Rv2660c, and evaluate its cellular immune response and protective efficacy against tuberculosis infection in BALB/c mice. Methods We constructed the p846 by using the cloning technology. The 6- to -8-week old female BALB/c mice were randomly divided into 4 groups: p846, pcDNA3.1, PBS and the BCG group. All mice were administrated intramuscularly with 50 μg recombinant plasmids at 0, 2, 4, 6 week. A single dose of BCG was injected subcutaneously in the BCG group. Two weeks after the final immunization, 10 mice in each group were used for cell proliferation, ELISPOT and FCM assay, BCG challenge experiment and HE staining of lung were performed at 4, 6 weeks later, respectively. Results The p846 vaccine could effectively induce the specific T cell proliferation(P < 0.001) and increase the numbers of IFN-γ+T cells(P <0.001), compared with those in the PBS group and the vector conreol group. The mouse lung tissue presented very mild lung inflammation in the p846 group, compared with other groups. Conclusion Vaccine p846 could not only induce strong cellular immune response, but also efficiently protect BALB/c mice against M.tb infection.
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