小鼠巨噬细胞源性趋化因子作为佐剂增强不可分型流感嗜血杆菌P6蛋白疫苗的免疫保护作用

Mouse macrophage-derived chemokine as an adjuvant enhances the protective effect of P6 protein vaccine of nontypeable Haemophilus influenzae

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 P6 protein of nontypeable Haemophilus influenzae（ NTHi） in prokaryotic cells and observe the immune effect of macrophage-derived chemokine（ MDC） as an adjuvant on NTHi-P6 protein vaccine. Methods The encoding sequence of NTHi-P6 protein was inserted into plasmid PGEX-6P2,and the recombinant plasmid PGEX-6P2 /P6 was transformed into E. coli XL1-Blue. In the expression system,the P6 protein was induced and expressed. BALB /c mice were randomized into three groups that were respectively inoculated intraperitoneally with P6 protein combined with Freund＇s adjuvant and MDC,P6 protein combined with Freund＇s adjuvant and only PBS,3 times at 0,14 and 28 days,respectively.Sera were collected from 12 mice of each group 14 days after each injection. The titer of IgG antibodies was detected by ELISA. Splenocytes were isolated from 3 mice of each group for determining the levels of IL-4 and IFN-γ by ELISA.Furthermore,the other 15 mice of each group were challenged with 10 × LD50 NTHi ATCC49247 to observe the protective effect of the vaccine. Results The IgG antibody could be induced by p6 protein combined with Freund＇s adjuvant and MDC and P6 protein combined with Freund＇s adjuvant,and the titers were 1 ∶ 1 140. 25 and 1 ∶ 3 044. 38,respectively. The differences in the antibody titers and the levels of IFN-γ between the two groups were significant statistically（ P 〈0. 05）.There was no difference in IL-4 levels between the two groups（ P〈 0. 05）. After challenged with 10 × LD50 NTHi ATCC49247,the survival rate of mice in P6 protein combined with Freund＇s adjuvant and MDC group was 80%,and significantly higher than that in the PBS group（ P 〈0. 05）,but no statistical difference was noted when compared with P6 protein combined with Freund＇s adjuvant group. Conclusion MDC as an adjuvant could enhance the immune protective effect of NTHi-P6 protein vaccine to an extent.