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目的评价Mtb复苏因子DNA疫苗(resuscitation-promoting factor DNA,Rpf-DNA)对Mtb感染宿主的免疫保护作用。方法构建复苏因子D(Rpf D-DNA)和复苏因子E(Rpf E-DNA)质粒疫苗,180只BALB/c小鼠(4周龄)分为6组,每组30只,分别用空质粒、Rpf D-DNA质粒、Rpf E-DNA质粒、BCG、生理盐水免疫,H37Rv标准株气溶胶感染。检测血清复苏因子(Rpf)抗体、IFN-γ;RpfD、E刺激淋巴细胞后进行淋巴细胞增殖、细胞杀伤实验;检测细胞上清IFN-γ、IL-12、IL-2;对肺组织进行病理学检测和细菌负荷(CFU)计数。结果 (1)疫苗免疫组血清Rpf D、Rpf E抗体水平:Rpf D组0.32±0.1,Rpf E组0.39±0.1,BCG组0.02±0.01,空质粒组0.01±0.0,生理盐水组0.09±0.04,空白对照组0.03±0.01,RpfD组与RpfE组抗体水平与BCG组、空质粒组、生理盐水组、空白对照组比较差异有显著统计学意义,F值分别为Rpf D:45.6,43.2,45.1,45.7;Rpf E:51.6,53.6,51.0,52.2;P值均<0.01。血清IFN-γ:Rpf D组(43.9±24.8)pg/ml,Rpf E组(45.9±21.0)pg/ml,BCG组(21.0±11.0)pg/ml,空质粒组(7.9±4.9)pg/ml,生理盐水组(4.7±2.1)pg/ml,空白对照组(5.8±4.7)pg/ml,Rpf D、Rpf E质粒组与BCG组比较差异有统计学意义(F值分别为Rpf D:10.2;Rpf E:12.4;P值均<0.05),与空质粒组、生理盐水组、空白对照组比较差异有显著统计学意义,F值分别为Rpf D:15.6,17.8,17.3;Rpf E:17.5,21.1,20.7;P值均<0.01。(2)淋巴细胞增殖实验CCK-8:Rpf D组176.0±4.2,Rpf E组183.0±4.3,BCG组101.0±1.1,空质粒组100.0±6.8,生理盐水组104.0±8.4,空白对照组116.0±2.2,RpfD、RpfE质粒组与BCG组、空质粒组、生理盐水组、空白对照组比较差异有统计学意义,F值分别为Rpf D:9.5,10.1,10.0,9.0;Rpf E:11.2,12.9,11.7,10.3;P值均<0.05。细胞杀伤实验:Rpf D组32.0±3.2,Rpf E组30.0±4.2,BCG组16.0±5.9,空质粒组3.3±1.5,生理盐水组6.7±0.5,空白对照组7.3±3.5,Rpf D、Rpf E质粒组与BCG组、空质粒组、生理盐水组、空白对照组比较差异有统计学意义,F值分别为Rpf D:8.8,14.5,13.7,11.9;Rpf E:8.1,12.5,11.6,11.1;P值均<0.05。(3)Rpf D、Rpf E蛋白各自刺激淋巴细胞后上清细胞因子检测:IL-2:Rpf D组9.5±2.4,RpfE组9.2±1.2,BCG组2.4±2.1,空质粒组1.2±0.3,生理盐水组1.8±1.0,空白对照组1.5±0.7,Rpf D、Rpf E质粒组与其他各组比较差异有统计学意义,F值分别为Rpf D:12.5,14.6,13.5,13.9;Rpf E:12.0,13.6,13.1,13.2;P值均<0.05。IFN-γ:Rpf D组22.2±5.7,Rpf E组28.7±14.4,BCG组16.1±10.1,空质粒组9.8±1.6,生理盐水组13.2±2.1,空白对照组15.7±2.9,RpfD、RpfE质粒组与其他各组比较差异有统计学意义,F值分别为Rpf D:7.8,12.6,8.7,8.3;Rpf E:11.3,16.4,14.7,14.2;P值均<0.05。结论实验表明复苏因子疫苗能诱导Mtb感染小鼠产生体液免疫和细胞免疫,有希望成为结核病候选疫苗之一。
Objective To evaluate the immunoprotective effect of Mtb resuscitation-promoting factor DNA (Rpf-DNA) on Mtb-infected host. Methods Plasmids of Rpf D-DNA and Rpf E-DNA were constructed. One hundred and eighty BALB / c mice (4 weeks old) were divided into 6 groups (n = 30) , Rpf D-DNA plasmid, Rpf E-DNA plasmid, BCG, saline, H37Rv standard aerosol infection. Serum recovery factor (Rpf) antibody and IFN-γ were detected; RpfD, E lymphocytes were stimulated with lymphocyte proliferation and cytotoxicity test; IFN-γ, IL-12 and IL- Neutrophil testing and bacterial load (CFU) counting. (1) The levels of Rpf D and Rpf E in serum of vaccine-immunized group were 0.32 ± 0.1 in Rpf D group, 0.39 ± 0.1 in Rpf E group, 0.02 ± 0.01 in BCG group, 0.01 ± 0.0 in empty plasmid group and 0.09 ± 0.04 in normal saline group, The blank control group was 0.03 ± 0.01. The antibody levels of RpfD group and RpfE group were significantly different from that of BCG group, empty plasmid group, normal saline group and blank control group. The F values were Rpf D: 45.6, 43.2, 45.1, 45.7; Rpf E: 51.6, 53.6, 51.0, 52.2; P values <0.01. Serum levels of IFN-γin Rpf D group (43.9 ± 24.8) pg / ml, Rpf E group (45.9 ± 21.0) pg / ml, BCG group (21.0 ± 11.0) pg / ml and empty plasmid group ml, 4.7 ± 2.1 pg / ml in normal saline group, 5.8 ± 4.7 pg / ml in blank control group, and Rpf D, Rpf E plasmid group were significantly different from BCG group (F values were Rpf D: 10.2; Rpf E: 12.4; P <0.05). There were significant differences between the control group and the empty plasmid group, the saline group and the blank control group. The values of F were Rpf D: 15.6,17.8,17.3; Rpf E: 17.5,21.1,20.7; all P <0.01. (2) Lymphocyte proliferation assay CCK-8: 176.0 ± 4.2 in Rpf D group, 183.0 ± 4.3 in Rpf E group, 101.0 ± 1.1 in BCG group, 100.0 ± 6.8 in empty plasmid group, 104.0 ± 8.4 in saline group and 116.0 ± in blank control group 2.2, RpfD, RpfE plasmid group and BCG group, empty plasmid group, saline group, blank control group, the difference was statistically significant, F values were Rpf D: 9.5,10.1,10.0,9.0; Rpf E: 11.2,12.9 , 11.7, 10.3; all P <0.05. Cell killing assay: Rpf D 32.0 ± 3.2, Rpf E 30.0 ± 4.2, BCG 16.0 ± 5.9, empty plasmid 3.3 ± 1.5, saline 6.7 ± 0.5, blank control 7.3 ± 3.5, Rpf D, Rpf E There was significant difference between plasmid group and BCG group, empty plasmid group, normal saline group and blank control group, F values were Rpf D: 8.8,14.5,13.7,11.9; Rpf E: 8.1,12.5,11.6,11.1; P <0.05. (3) Rpf D and Rpf E respectively stimulated lymphocytes to up-regulate cytokines after IL-2: Rpf D group 9.5 ± 2.4, RpfE group 9.2 ± 1.2, BCG group 2.4 ± 2.1, empty plasmid group 1.2 ± 0.3, 1.8 ± 1.0 in normal saline group and 1.5 ± 0.7 in blank control group. There was significant difference between Rpf D and Rpf E plasmid group and other groups, F values were Rpf D: 12.5,14.6,13.5,13.9, Rpf E: 12.0, 13.6, 13.1 and 13.2 respectively; all P <0.05. IFN-γ: 22.2 ± 5.7 in Rpf D group, 28.7 ± 14.4 in Rpf E group, 16.1 ± 10.1 in BCG group, 9.8 ± 1.6 in empty plasmid group, 13.2 ± 2.1 in saline group, 15.7 ± 2.9 in blank control group, Compared with other groups, the difference was statistically significant, F values were Rpf D: 7.8,12.6,8.7,8.3; Rpf E: 11.3,16.4,14.7,14.2; P values were <0.05. Conclusion Experiments show that the recovery factor vaccine can induce humoral and cellular immunity in mice infected with Mtb, and hopefully become one of the candidate vaccine for tuberculosis.