Ag85B DNA/H37Ra序贯免疫效果的探讨

Immune efficacy of Ag85B DNA/H37Ra prime-boost vaccination regimen for tuberculosis in mice

目的:初步探讨含结核分枝杆菌Ag85B成熟蛋白的DNA(pTB30m)和结核菌H37Ra序贯免疫小鼠产生的特异性免疫应答。方法:重组质粒pTB30m用碱裂解法制备后进行质量鉴定。用pTB30m肌注初次免疫小鼠2周后,H37Ra皮内加强免疫作为DNA-85B/H37Ra组,同时设定DNA-85B/BCG组、H37Ra组、BCG组及未免疫组。免疫4周或8周后,用ELISA法检测小鼠血清中抗PPD IgG抗体的水平和MTT法检测其脾淋巴细胞的刺激指数(SI)。结果:酶切鉴定pTB30m所含外源基因片段大小正确,并且纯度较高。DNA-85B/H37Ra组小鼠血清中抗PPD IgG水平、脾淋巴细胞的SI均显著高于未免疫组(P<0.05);其抗PPD IgG水平稍高于DNA-85B/BCG组、H37Ra组及BCG组,但它们之间无显著性差异(P>0.05);其脾淋巴细胞的SI显著高于H37Ra、BCG组(P<0.05),而与DNA-85B/BCG组比较,仅在4周有显著性差异。在DNA-85B/H37Ra组内,SI在4周显著高于8周(P<0.05),而血清中抗PPD IgG水平8周均显著高于4周(P<0.05)。结论:DNA-85B/H37Ra序贯免疫策略可以诱导小鼠产生特异性体液免疫和细胞免疫,初步证明其免疫效果略优于BCG。

Objective：To investigate the immune efficacy after sequential immunization with Mycobacterium tuberculosis DNA vaccine encoding mature form of Ag85B（pTB30m）and Mycobacterium tuberculosis H37Ra in mice.Methods：Much of highly pure plasmid DNA （pTB30m）extracted by alkaline lysis method was confirmed by restriction endonuclease digestion.Then,its DNA concentration and purity were determined by UV spectrophotometry. At various intervals （4weeks, 8weeks）after sequential immunization, ELISA was used to detect the level of the serum antibody against PPD. Also,the spleen lymphocytes of mice were cultured with PPD in vitro. Lymphocyte transformation was detected by MTT assay. Results：Prepared pTB30m was highly pure and came to the needed concentration. Compared with Group Naive control, the specific antibody levels against PPD and the stimulation index （SI）of spleen lymphocytes were all statistically higher in Group DNA-85B/H37Ra （P〈0.05）, but the specific antibody levels were not significant different（P〉0.05）,as compared with Group DNA-g5B/BCG,Group H37Ra and Group BCG, However,compared with Group H37Ra and Group BCG, the SI of mice was significantly larger in Group DNA-85B/H37Ra （P〈0.05）,but compared with Group DNA-85B/BCG,it was only statistically higher at 4weeks but not at 8 weeks. Simultaneously,in Group DNA-85B/H37Ra,the SI of the spleen lymphocytes was much higher at 4weeks than that at 8weeks（P〈0.05）, but the specific antibody levels were much higer at 8weeks than those at 4weeks （P〈0.05）.Conclusion：The DNA-85B/H37Ra regimen for tuberculosis prevention in mice stimulate the specific antibody and induce cellular immunity.The immune efficacy of the DNA-85B/H37Ra regimen is slighterly better than that of BCG.