Han Xiaoyan, Wang Chenhong, Qiao Haixia, Zhang Yanxia, Chang Yueli, Zhang Yutuo
Department of Microbiology, Hebei North University, Zhangjiakou 075000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Sep;30(9):913-6.
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.
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.
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.
MDC as an adjuvant could enhance the immune protective effect of NTHi-P6 protein vaccine to an extent.
在原核细胞中表达不可分型流感嗜血杆菌（NTHi）的P6蛋白，并观察巨噬细胞衍生趋化因子（MDC）作为佐剂对NTHi - P6蛋白疫苗的免疫效果。
将NTHi - P6蛋白的编码序列插入质粒PGEX - 6P2，构建重组质粒PGEX - 6P2/P6，并转化至大肠杆菌XL1 - Blue中。在该表达系统中诱导表达P6蛋白。将BALB/c小鼠随机分为三组，分别于0、14和28天腹腔注射P6蛋白联合弗氏佐剂和MDC、P6蛋白联合弗氏佐剂和仅注射PBS，每组各12只小鼠。每次注射后14天采集血清，用ELISA法检测IgG抗体效价。每组取3只小鼠分离脾细胞，用ELISA法测定IL - 4和IFN - γ水平。此外，每组另取15只小鼠用10×LD50的NTHi ATCC49247进行攻毒，观察疫苗的保护效果。
P6蛋白联合弗氏佐剂和MDC以及P6蛋白联合弗氏佐剂均能诱导产生IgG抗体，效价分别为1:1 140.25和1:3 044.38。两组抗体效价及IFN - γ水平差异有统计学意义（P<0.05）。两组IL - 4水平差异无统计学意义（P>0.05）。用10×LD50的NTHi ATCC49247攻毒后，P6蛋白联合弗氏佐剂和MDC组小鼠存活率为80%，显著高于PBS组（P<0.05），但与P6蛋白联合弗氏佐剂组相比无统计学差异。
MDC作为佐剂能在一定程度上增强NTHi - P6蛋白疫苗的免疫保护效果。
