结核分枝杆菌ESAT6-Fc DNA疫苗的免疫效应评价

Evaluation of immune effect of ESAT6-Fc DNA vaccine against Mycobacterium tuberculosis

目的 评价结核分枝杆菌ESAT6-Fc DNA疫苗的免疫效应,为下一步疫苗抗M.tb感染的保护性试验奠定基础。方法 将雌性C57BL/6J小鼠随机分为2组,每组5只,分别经大腿肌内注射含HSV2gD信号肽基因、Rv3875基因和小鼠Fc基因(共1 116 bp核苷酸)的重组质粒pcD-ESAT6-Fc及PBS,剂量均为50μg/条腿,共100μg。初次免疫后每周免疫1次,连续免疫3次。初次免疫后14、28、42 d采血,分离血清,采用间接ELISA法检测血清中IgG、IgG1及IgG2a抗体效价;采集初次免疫后42 d的血清及脾脏,分离脾细胞,采用流式细胞术检测分泌IFNγ的CD4^(+)及CD8^(+)T细胞比例,ELISA法检测血清中IFNγ和IL-2含量。结果 初次免疫后14 d,重组质粒免疫组小鼠血清中即可检测到特异性IgG、IgG1及IgG2a抗体,且抗体效价持续升高;初次免疫后42 d,重组质粒免疫组CD3^(+)CD4^(+)IFNγ^(+)和CD3^(+)CD8^(+)IFNγ^(+)细胞比例以及IFNγ和IL-2含量均显著高于PBS组(t分别为12.43、13.35、7.04和6.65,P均<0.001)。结论 重组质粒pcDESAT6-Fc可有效诱导小鼠产生体液免疫和细胞免疫应答,为ESAT6-Fc DNA疫苗的进一步研制奠定了实验基础。

Objective To evaluate the immune effect of ESAT6-Fc DNA vaccine against Mycobacterium tuberculosis(M.tb),so as to lay a foundation of the next protective test of the vaccine against M.tb infection.Methods The female C57BL/6J mice were randomly divided into two groups with 5 mice in each group,which were injected with the recombinant plasmid pcD-ESAT6-Fc containing HSV2gD signal peptide gene,Rv3875 gene and mouse Fc gene(1116 bp nucleotides)as well as PBS intramuscularly through thigh,respectively,with the dosage of 50μg/leg,100μg in total.After the first immunization,immunization was boosted once a week for three times continuously.Blood samples were collected 14,28,42 d after the first immunization,and the titers of IgG,IgG1 and IgG2a in serum were detected by indirect ELISA;Serum and spleen samples were collected 42 d after the first immunization,spleen cells were isolated and detected for the ratio of CD4~+and CD8~+T cells secreting IFNγby flow cytometry,and the contents of IFNγand IL-2 in serum were determined by ELISA.Results IgG,IgG1 and IgG2a specific antibodies were detected in the serum of mice immunized with the recombinant plasmid 14 d after the first immunization,and the titers continued to increase;42 d after the first immunization,The ratio of CD3^(+)CD4^(+)IFNγ^(+)and CD3^(+)CD8^(+)IFNγ^(+)cells and the contents of IFNγand IL-2 in the recombinant plasmid immunized group were significantly higher than those in the PBS group(t=12.43,13.35,7.04 and 6.65,respectively,each P<0.001).Conclusion The recombinant plasmid pcD-ESAT6-Fc can effectively induce the humoral and cellular immune responses in mice,which lays an experimental foundation for the further development of ESAT6-Fc DNA vaccine.+cells and the contents of IFNγand IL-2 in the recombinant plasmid immunized group were significantly higher than those in the PBS group(t=12.43,13.35,7.04 and 6.65,respectively,each P<0.001).Conclusion The recombinant plasmid pcD-ESAT6-Fc can effectively induce the humoral and cellular immune responses in mice,which lays an experimental foundation for the further development of ESAT6-Fc DNA vaccine.