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目的用原核细胞表达不可分型流感嗜血杆菌(NTHi)P6蛋白并观察巨噬细胞源性趋化因子(MDC)对NTHi-P6蛋白疫苗免疫效果的影响。方法构建原核表达质粒PGEX-6P2/P6,转化E.coli XL1-Blue,诱导P6蛋白的表达。将BALB/c小鼠随机分为P6蛋白联合Freund佐剂与MDC组、P6蛋白联合Freund佐剂组、PBS对照组。分别于0、14、28 d经腹腔免疫,末次免疫后14 d,每组取12只小鼠取血,ELISA检测血清中IgG抗体水平。每组取3只小鼠,制备脾淋巴细胞,ELISA检测IL-4和IFN-γ水平。用10×LD50NTHi攻击每组剩余15只小鼠,观察免疫保护作用。结果 P6蛋白联合Freund佐剂与MDC组、P6蛋白联合Freund佐剂组均能诱导小鼠产生IgG抗体,其滴度分别为1∶1 140.25、1∶3 044.38,P6蛋白联合Freund佐剂与MDC组小鼠抗体滴度和IFN-γ水平明显高于P6蛋白联合Freund佐剂组(P<0.05),但IL-4水平2组间差异无统计学意义(P>0.05)。经NTHi攻击后,P6蛋白联合Freund佐剂与MDC组小鼠的生存率达80%,与PBS组相比差异有统计学意义(P<0.05),但与P6蛋白联合Freund佐剂组相比无显著性差异。结论 MDC作为蛋白佐剂,在一定程度上增强了NTHi-P6蛋白疫苗的免疫保护作用。
Objective To express PTH protein of NTHi in prokaryotic cells and observe the effect of macrophage-derived chemotactic factor (MDC) on the immunogenicity of NTHi-P6 protein vaccine. Methods The prokaryotic expression plasmid PGEX-6P2 / P6 was constructed and transformed into E. coli XL1-Blue to induce P6 protein expression. BALB / c mice were randomly divided into P6 protein combined with Freund adjuvant and MDC group, P6 protein combined Freund adjuvant group and PBS control group. The mice were immunized intraperitoneally on day 0, day 14 and day 28 respectively. At 14 d after the last immunization, 12 mice in each group were sacrificed and their serum IgG levels were measured by ELISA. Spleen lymphocytes were prepared from 3 mice in each group, and the levels of IL-4 and IFN-γ were detected by ELISA. The remaining 15 mice in each group were challenged with 10 x LD50 NTHi and the immunoprotection was observed. Results P6 protein combined with Freund’s adjuvant and MDC group, P6 protein and Freund’s adjuvant group all could induce IgG antibody production in mice with the titer of 1:1 140.25,1:3 044.38, P6 protein combined with Freund adjuvant and MDC The antibody titers and IFN-γ levels of the mice were significantly higher than those of P6 and Freund adjuvant (P <0.05), but there was no significant difference between the two groups (P> 0.05). After NTHi challenge, the survival rate of P6 protein combined with Freund adjuvant and MDC mice was 80%, which was significantly different from PBS group (P <0.05), but compared with P6 protein and Freund adjuvant group No significant difference. Conclusion MDC as a protein adjuvant, to some extent, enhances the immunoprotection of NTHi-P6 protein vaccine.