不可分型流感嗜血杆菌P6 DNA疫苗与蛋白疫苗序贯免疫效应研究

Study of immune responses to nontypeable Haemophilus influenzae by P6 DNA prime protein-boost immunity strategy

观察不可分型流感嗜血杆菌P6DNA疫苗初免蛋白疫苗加强免疫方式的免疫效果。大量扩增pcDNA3-P6。将65只BALB/c小鼠随机分PBS对照组、pcDNA3.1对照组、pcDNA3.1-P6组、P6蛋白组、pcDNA3.1-P6/P6蛋白组,分别于0、14、28d免疫。质粒每次每只接种100μg,蛋白每次每只接种10μg。末次免疫后14d,每组取10只小鼠取血,ELISA检测血清中特异性IgG抗体滴度。每组取3只小鼠处死,制备脾淋巴细胞,ELISA检测IL-4和IFN-γ水平,CCK-8法检测脾淋巴细胞增殖指数。用15LD50NTHi攻击每组剩余10只小鼠,观察免疫保护作用。结果:pcDNA3.1-P6/P6蛋白组小鼠的脾淋巴增殖指数和IFN-γ水平明显高于质粒组和蛋白组(P<0.05)。pcDNA3.1-P6/P6蛋白组小鼠IgG抗体滴度、IL-4水平和动物生存率明显高于质粒组(P<0.05)但和蛋白组相比无明显差异(P>0.05)。因此pcDNA3.1-P6初免P6蛋白加强免疫的方式可以提高疫苗的免疫效果。

The purpose of this study was to observe the efficacy of a vaccination for haemophilus influenza based on a DNA prime- protein boostier strategy. Sixty-five BALB/c mice were randomly divided into 5 groups and inoculated respectively with PBS, pcDNA3.1, pcDNA3.1-P6,P6 protein, or pcDNA3.1-P6/P6 protein for 3 times at day 0, 14 and 28. pcDNA3-P6 was amply amplified. Sixty-five BALB/c mice were randomly divided into 5 groups and then inoculated respectively with PBS, pcD- NA3.1, pcDNA3.1-P6,P6 protein, or pcDNA3. 1-P6/P6 protein for 3 times at day 0, 14 and 28. The immunizing dose of plasmid pcDNA3.1-P6 was 100 μg and that of protein P6 was 10 μg. At day 14 after the last inoculation, sera were collected from 10 mice per group and titers of IgG antibodies were detected by ELISA . In addition, splenocytes from 3 mice per group were isolated and cultured to monitor cell proliferation with CCK-8 kit and the production of IL-4 and IFN-γ was detected by ELISA. Another 10 mice per group were challenged with 15LDs0 of haemophitus influenzae ATCC49247 to observe the protec- ting effect of the vaccines. We found that pcDNA3. 1-P6/P6 group produced higher specific proliferation index and levels of IFN-γ than those of mice immunized with either pcDNA3, 1-P6 or P6 protein （P〈0 . 05） alone after the third immunization. The titers of specific IgG, IL-4 and the survival rate were significantly higher in pcDNA3.1-P6/P6 group than those in the pcD- NA3. 1-P6 group （P〈0 . 05）, but there was no statistically difference when compared with P6 protein group （P〈0 . 05）. Therefore, combined application of P6 DNA prime protein-booster regimen effectively induced stronger immune responses than either plasmid or P6 protein alone.