Rpf结构域及其突变体对MTB感染小鼠的免疫治疗作用  

Therapeutic Efficacy of Micrococcus Luteus Rpf Domain and its Mutants against Mycobacterium Tuberculosis in Mice

作　　者：尚淑琴[1] 赵勇[2] 赵善民[2] 张彩琴[2] 师长宏[2] 

机构地区：[1]西安市疾病预防控制中心,陕西西安710054 [2]第四军医大学实验动物中心,陕西西安710032

出　　处：《现代生物医学进展》2013年第6期1048-1051,共4页Progress in Modern Biomedicine

基　　金：陕西省自然科学基金项目(2010JM4012)

摘　　要：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 investigate therapeutic efficacy of Micrococcus luteus Rpf domain and its mutants E54K （Rpfdl）, E54A （Rpfd2） and E54K＋D48A （Rpfd3） against Mycobacterium tuberculosis infection mice. Methods： Recombinant M.luteus Rpf domain （Rpfd） and its mutants E54K （Rpfdl）, E54A （Rpfd2）, E54K ＋ D48A （Rpfd3） were expressed in E.coli BL21 and purified by GST affinity chromatography. BALB/c mice were infected with the H37Rv strain via the tail vein at a dose of 105 CFU/mouse. Four weeks after infected, the mice were randomly divided into six groups. Four groups were treated with the purified protein Rpfd, Rpfdl, Rpfd2 and Rpfd3, respectively, three times with 2-week intervals. The drug treated group was co-administered isoniazid （INH, 54.25 mg/L） with rifampicin （RFP, 52.5 mg/L）. The mice in the control group received only saline （negative control）. At 13 weeks post-infection Determination of bacterial burden in lung and histopathology were performed. Results： The expressed proteins can specific bind with anti- Rpfd mAb, showed a 32 kDa band. At 11 weeks and 13 weeks post-infection, the bacterial burden of lungs in mice that received Rpfd, Rpfdl and Rpfd2 is significantly lower than that in the saline treated group （P〈0.05） and Rpfd3 group, but not significantly effective compared with that in the 1NH＋RFP treated group. Conclusion： After infected by MTB, the mice were treated by Rpfd protein immune therapy. And the treatment result showed that Rpfd, mutant Rpfdl and Rpfd2 protein can significantly reduce the lung load count, and induced lymphocyte value-added, and provides a theoretical basis and experimental basis for the preparation of MTB immune treatment vaccine by using Rpf structure domain mutant.

关 键 词：结核分支杆菌 RPF蛋白 突变体 免疫治疗 结构域 

分 类 号：Q95-3[生物学—动物学] R378.911[医药卫生—病原生物学]