Al(OH)_(3)和CpG寡核苷酸免疫佐剂对结核分枝杆菌Ag85B蛋白免疫原性的影响

Effects of Al(OH)_(3) and CpG oligodeoxynucleoties adjuvants on the immunogenicity of Mycobacterium tuberculosis Ag85B protein

目的 探究Al(OH)_(3)和CpG寡核苷酸免疫佐剂对结核分枝杆菌(Mycobacterium tuberculosis,Mtb)Ag85B蛋白免疫原性的影响。方法 将Mtb Ag85B蛋白在大肠埃希菌中进行重组表达,经亲和层析纯化后分别与Al(OH)_(3)、CpG寡核苷酸(简称CpG)及Al(OH)_(3)和CpG组成的复合佐剂[Al(OH)_(3)+CpG]配伍,经皮下免疫BALB/c小鼠,初免后28 d加强免疫1次,分别于初免后14、42和56 d采集免疫血清,采用ELISA检测血清中Ag85B特异性IgG、lgG1、IgG2a和IgG2b水平;初免后42 d,每组取4只小鼠无菌分离脾淋巴细胞,利用酶联免疫斑点检测各组细胞中分泌γ干扰素和白介素4的淋巴细胞数量,采用流式细胞术检测各组细胞中CD4^(+)T和CD8^(+)T淋巴细胞的比例。结果 1.5μg或4.5μg Ag85B重组蛋白与Al(OH)_(3)+CpG配伍免疫后均诱导了高水平的Ag85B特异性IgG,免疫效果优于该蛋白与2种佐剂单独配伍组。1.5μg或4.5μg Ag85B重组蛋白与Al(OH)_(3)+CpG配伍免疫血清中IgG亚型为IgG1、IgG2a和IgG2b混合型,其中IgG2a和IgG2b水平均高于Al(OH)_(3)配伍免疫组,差异均有统计学意义(P均<0.001)。酶联免疫斑点检测显示,4.5μg Ag85B重组蛋白与Al(OH)_(3)+CpG配伍免疫小鼠的脾淋巴细胞中分泌γ干扰素和白介素4的淋巴细胞数量均高于Al(OH)_(3)配伍免疫组,差异均有统计学意义(P均<0.05);流式细胞术检测显示,1.5μg或4.5μg Ag85B重组蛋白与Al(OH)_(3)+CpG配伍免疫小鼠的T淋巴细胞中CD4^(+)T淋巴细胞的占比均高于Al(OH)_(3)配伍免疫组。结论 Al(OH)_(3)和CpG联合使用可能协同增强Ag85B重组蛋白的免疫原性。

Objective To investigate the effects of Al(OH)_(3)and CpG oligodeoxynucleoties adjuvants on the immunogenicity of Mycobacterium tuberculosis(Mtb)Ag85Bprotein.MethodsAg85B protein of Mycobaclerium tuberculosiswas expressed in E.coli and purified by affinity chromatography.Then,BALB/c mice were immunized subcutaneously with the purified Ag85B protein formulated with Al(OH)_(3),CpG oligodeoxynucleoties(CpG for short),or compound adjuvant of Al(OH),and CpG(Al(OH)_(3)+CpG)respectively and booster immunization 28 days later.Serum samples were collected at 14,42 and 56 days after the primary vaccination.ELISA was used to detect the levels of Ag85B-specific IgG,IgG1,IgG2a and IgG2b.Forty-two days after primary immunization,spleen lymphocytes were aseptically isolated from 4 mice in each group.The number of interferon-γ(IFN-γ)and interleukin-4(IL-4)secreting lymphocytes in each group was detected by enzyme-linked immunosorbent spot,and the proportion of CD4^(+)and CD8^(+)T lymphocytes in each group was detected by flow cytometry.Results High levels of Ag85B-specific IgG were induced by immunization with 1.5μg or 4.5μg of recombinant Ag85B protein formulated with Al(OH)_(3)+CpG,which was better than the protein formulated with either adjuvant alone.The IgG subclasses in mice immunized with 1.5μg or 4.5μg recombinant Ag85B protein formulated with Al(OH)_(3)+CpG were IgG1,IgG2a and IgG2b,and the levels of IgG2a and IgG2b were higher than those of the same protein formulated with Al(OH)_(3),exhibiting statistically significant difference(all P<0.001).Enzyme-linked immunosorbent spot showed that the number of spleen lymphocytes secreting IFN-γ and IL-4 in mice immunized with 4.5μg recombinant Ag85B protein with Al(OH)_(3)+CpG was higher than those of the same protein formulated with Al(OH)_(3),exhibiting statistically significant difference(both P<0.05).Flow cytometry showed that the proportion of CD4^(+)T lymphocytes in T lymphocytes of mice immunized with 1.5μg or 4.5μg recombinant Ag85B protein formulated with Al(OH)_(3)+CpG was higher than that of the same protein formulated with Al(OH)_(3).Conclusion The combination of Al(OH)_(3) and CpG may enhance the immunogenicity of recombinant Ag85B protein.