结核分枝杆菌Ag85B-Fc2a DNA疫苗的免疫原性及免疫保护性评价

Immunogenicty and immunoprotective effect of Mycobacterium tuberculosis Ag85B-Fc2a DNA vaccine

目的 评价结核分枝杆菌(Mycobacterium tuberculosis,M.tb)Ag85B-Fc2a DNA疫苗在小鼠体内产生的免疫原性及免疫保护性,以期为该疫苗的研发提供实验依据。方法 将重组质粒pcD-Ag85B-Fc2a经双酶切和测序鉴定后,转染CHO-K1细胞,采用Western blot法检测融合蛋白Ag85B-Fc2a的表达。将载体pcDNA3.1(+)及重组质粒pcD-Ag85BFc2a经大腿肌内分别注射雌雄C57BL/6J小鼠(对照组及免疫组),每组10只,初次免疫后2周进行加强免疫。初次免疫后14、28及42 d,经小鼠眼眶采血,分离血清,采用间接ELISA法检测血清中IgG抗体效价;初次免疫后42 d,无菌取小鼠脾脏,制备为单细胞悬液,采用流式细胞术检测CD4^(+)及CD8^(+)T细胞比例;初次免疫后42 d,经尾静脉注射M.tb H37Ra,10~6 CFU/只,攻毒28 d后,断颈处死小鼠,无菌分离小鼠肺脏及脾脏,采用平板法检测左侧肺脏及脾脏荷菌数,金胺O荧光染色法检测右侧肺脏的荷菌情况。结果 经双酶切及测序鉴定,重组质粒pcD-Ag85B-Fc2a构建正确,转染CHO-K1细胞后,可检测到相对分子质量约70 000的融合蛋白Ag85B-Fc2a。初次免后14、28、42d,免疫组小鼠血清Ag85B特异性IgG抗体效价分别为1:3 200、1:12 160、1:12 800,对照组小鼠血清中均未检测到抗体效价。初次免疫后42 d,对照组及免疫组小鼠脾脏中CD4+T细胞比例分别为(23.61±0.64)%及(26.92±0.80)%,CD8^(+)T细胞比例分别为(14.12±0.87)%及(18.78±0.94)%,差异均有统计学意义(t分别为3.23和3.64,P均<0.05)。M.tb H37Ra感染28 d,对照组及免疫组小鼠左侧肺脏荷菌数分别为(4.73±0.13)及(3.81±0.14) CFU,脾脏荷菌数分别为(5.02±0.19)及(4.30±0.13) CFU,差异均有统计学意义(t分别为4.65和3.12,P均<0.01);右侧肺脏荷菌情况与左侧一致。结论 Ag85B-Fc2a DNA疫苗在小鼠体内可诱导特异性体液及细胞免疫效应,对M.tb H37Ra感染可产生良好的保护力。

Objective To evaluate the immunogenicity and protective effect of Mycobacterium tuberculosis(M.tb) Ag85B-Fc2a DNA vaccine in mice,so as to provide experimental basis for the development of the vaccine.Methods The recombinant plasmid pcD-Ag85B-Fc2a was identified by double digestion and sequencing,and then transfected into CHO-K1 cells.The expression of fusion protein Ag85B-Fc2a was detected by Western blot.Vector pcDNA3.1(+) and recombinant plasmid pcDAg85B-Fc2a were injected into female C57BL/6J mice through thigh muscle respectively(control group and immunization group),with 10 mice in each group,and booster immunization was carried out two weeks after the initial immunization.At14 d,28 d and 42 d after the initial immunization,serum samples was separated,and the titers of IgG antibody in the serum were detected by indirect ELISA.At 42 d after the initial immunization,the spleen of mice was taken aseptically and made into single cell suspension,and the proportions of CD4~+and CD8~+T cells were detected by flow cytometry.The remaining mice were injected with M.tb H37Ra through the tail vein 42 d after the initial immunization at a dose of 10~6 CFU/mouse.After 28 d of challenge,the lung and spleen of mice were collected aseptically.The number of bacteria in the left lung and spleen was measured by plate method,and the bacteria in the right lung was detected by auramine O fluorescence staining.Results Double digestion and sequencing results showed that the recombinant plasmid pcD-Ag85B-Fc2a was constructed correctly.After transfection into CHO-K1 cells,the fusion protein Ag85B-Fc2a with a relative molecular mass of about 70 000was detected.The Ag85B-specific IgG antibody titer in serum of mice in the immunization group was 1:3 200,1:12 160,and 1:12 800 at 14 d,28 d,and 42 d after the initial immunization,respectively,but no antibody titer was detected in the serum samples of control group.At 42 d after the initial immunization,the percentages of CD4~+ T cells in mouse spleen of control group and immunization group were(23.61±0.64)% and(26.92±0.80)%,and the percentages of CD8~+T cells were(14.12±0.87)% and(18.78±0.94)%,respectively,with significant differences(t=3.23 and 3.64,respectively,each P <0.05).After infection with M.tb H37Ra for 28 d,the numbers of bacteria were(4.73±0.13) and(3.81±0.14)CFU in the left lung,and(5.02±0.19) and(4.30±0.13) C.FU in the spleen of control group and immunization group,respectively,with significant differences(t=4.65 and 3.12,respectively,each P <0.01).The bacteria loading in the right lung was consistent with that in the left lung.Conclusion Ag85B-Fc2a DNA vaccine can induce specific humoral and cellular immune effects in mice,and can produce good protective effect against M.tb H37Ra infection.