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目的:观察复苏促生长因子Rpf结构域(Rpfd)及其突变体E54K(Rpfd1),E54A(Rpfd2)和E54K+D48A(Rpfd3)对MTB感染小鼠的免疫治疗作用。方法:表达、纯化Rpfd及其突变体Rpfd1,Rpfd2和Rpfd3。结核毒株H37R v从尾静脉感染BALB/c小鼠,剂量为1×105C F U。感染4w后,随机分为6组,在感染后4w、6w和8周分别腹部皮下注射含50μg/ml相应蛋白Rpfd、Rpfd1、Rpfd2、Rpfd3的生理盐水,同时设异烟肼(INH,54.25 mg/L)联合利福平(RFP,52.5 mg/L)治疗组。在感染后11w和13w,分别取一侧肺脏计数荷菌数。将各时间点的感染小鼠另一侧肺脏固定后进行组织学观察。结果:表达产物均可与抗-Rpf结构域单抗特异性结合,相对分子量约为32 kDa。感染后11w和13w,Rpfd、Rpfd1、Rpfd2治疗组肺部荷菌量明显少于Rpfd3治疗组和生理盐水组(P<0.05),但其对肺部MTB的控制未达到INH+RFP联合治疗的效果(P<0.05)。结论:MTB感染小鼠后,利用Rpfd蛋白进行免疫治疗,发现Rpfd及其突变体Rpfd1和Rpfd2蛋白能够显著的降低肺脏荷菌数,并且诱导淋巴细胞的增值。为利用Rpf结构域突变体制备MTB的免疫治疗疫苗提供了理论依据和实验基础。
OBJECTIVE: To observe the immunotherapeutic effects of Rpf (Rpf) and its mutants E54K (Rpfd1), E54A (Rpfd2) and E54K + D48A (Rpfd3) on MTB-infected mice. Methods: Rpfd and its mutants Rpfd1, Rpfd2 and Rpfd3 were expressed and purified. BALB / c mice were infected with the H37R v strain of tuberculosis at a dose of 1 × 10 5 CFU from the tail vein. After infection for 4 weeks, the rabbits were randomly divided into 6 groups. Normal saline (50μg / ml), Rpfd, Rpfd1, Rpfd2 and Rpfd3 were injected subcutaneously at 4w, 6w and 8weeks respectively. / L) combined rifampicin (RFP, 52.5 mg / L) treatment group. 11w and 13w after infection, respectively, take one side of the lung count bacteria count. The lungs on the other side of infected mice at each time point were fixed for histological observation. Results: The expressed product could specifically bind to anti-Rpf domain mAb with a molecular weight of about 32 kDa. After 11 and 13 weeks of infection, the lungs in Rpfd, Rpfd1 and Rpfd2 groups were significantly less than those in Rpfd3 group and saline group (P <0.05), but their lung MTB control did not reach the level of INH + RFP combined treatment Effect (P <0.05). CONCLUSION: After immunotherapy with Rpfd protein in mice infected with MTB, Rpfd and its mutants Rpfd1 and Rpfd2 protein can significantly reduce the number of lung bacteria and induce lymphocyte proliferation. It provides theoretical basis and experimental basis for preparing immunotherapy vaccine of MTB with Rpf domain mutants.