ICAM-1编码基因对乙型脑炎DNA疫苗树突状细胞功能的影响

Effect of ICAM-1 genetic adjuvant on dendritic cell function of plasmid DNA encoding prME protein derived from Japanese enaphalitis virus

目的:研究细胞间粘附分子1(ICAM-1)编码基因对日本脑炎(JE)DNA疫苗脾脏树突状细胞功能的影响。方法:套式RT-PCR法获取BALB/c鼠ICAM-1编码基因,构建重组子pJME/ICAM-1和pICAM-1,脂质体法转染上述质粒于CHO细胞,Western blot法检测转染的CHO细胞中目的蛋白表达。实验分5组,包括:pJME/ICAM-1、pJME+PICAM-1、pJME、JE灭活疫苗和pcDNA3.1(+)免疫组,以不同免疫原肌注免疫BALB/c小鼠,流式细胞仪检测经不同免疫原免疫鼠后脾脏DC表型、抗原吞噬功能以及混合淋巴细胞反应。结果:融合表达重组质粒pJME/ICAM-1和单质粒pICAM-1经鉴定构建正确。pJME/ICAM-1组CD11c+CD86+DC和CD11c+ICAM-1+DC比例分别为(6.92±1.40)%、(7.18±0.57)%,高于其它免疫组(均P<0.05);pJME/ICAM-1和pJME+pICAM-1组DC表面CD80和MHCⅡ表达水平比较差异无统计学意义(P>0.05);pJME/ICAM-1与pJME+pICAM-1组内吞能力明显增强,平均荧光强度分别为437.11±47.60、416.67±29.12,显著高于其它组(P<0.05)。比较不同免疫原对细胞分裂的作用,以pJME/ICAM-1组最强,(73.69±7.32)%CD4+T细胞发生分裂,(45.40±2.57)%CD8+T细胞发生分裂,显著高于对照组(P<0.05)。结论:ICAM-1编码基因能够促进JE DNA疫苗脾脏树突状细胞的成熟,能够提供独立或放大B7分子的协同刺激信号。

Objective：To study the effect of intercellular adhesion molecule-1 （ICAM-1）coding gene on dendritic cell function induced by Japanese encephalitis （JE） virus DNA vaccine. Methods：ICAM-1 coding gene was amplified by nested-reverse tran- scriptase-polymerase chain reaction（ RT-PCR） technique from BALB/c murine lung tissue. Recombinant plasmids pJME/ICAM-1 and pICAM-1 were constructed by JE virus （JEV） prM-E protein with ICAM-l coding gene or ICAM-1 coding gene only, respectively. The plasmids were transfected into China hamster ovary （CHO） cells by Lipofeetamine2000. The coding protein expressions was analyzed by Western blot. For the i. m. immunization, the BALB/e mice were vaccinated with indicated immunogens with or without ICAM-1 gene. The changes of T lymphocyte subsets in the spleen and dendritic cell （ DC ） function, such as phenotype, antigen phagocytosis and the mixed lymphocyte reaction of splenic DC from mice immunized with different immunogens were evaluated by flow cytometry. The eyto- toxicity T lymphocyte（CTL） activity was assessed by lactate dehydrogenase （LDH）. Results：The constructed recombinant pICAM-1 and pJME/ICAM-1 were confirmed by restrict enzyme digestion and DNA sequencing. The percentage of CDllc＋ CD86 ＋ DCs and CD11c＋ ICAM-1 ＋ DC in the spleen in the pJME/ICAM-1 vaccinated groups were respectively （6. 92 ± 1. 40）% and （7. 18 ± 0. 57 ）% , higher than that in the pJME, JE inactivated vaccine and peDNA3.1 （ ＋ ） vaccinated group（P 〈 O. 05 ）, there was statisti- cally significant difference between pJME/ICAM-1 and pJME ＋ pICAM-l vaccinated groups （ P 〈 O. 05 ）. There was no significant difference in the CDS0 and MHC Ⅱ expression of DC surface between pJME/ICAM-1 and pJME ＋ pICAM-l group（P 〉0.05）. pJME/ ICAM-1 and pJME ＋ pICAM-1 groups markedly enhanced endocytosis of DCs（ MFI was separately 437.11 ±47.60 and 416.67 ± 29. 12） compared with other groups（P 〈 0.05）. The percentage of CD4 ＋T and CD8 ＋T cells division in spleen cells was respectively（73.69 ±7. 32）% and （45.40 ± 2. 57）% in the pJME/ICAM-1 vaccinated group, which significantly increased the division capacity of CD4 ＋ T and CD8 ＋ T cell compared with other groups（ P 〈 0.05 ）. The adjuvant effects of the ICAM-1 DNA on the cellular immune responses induced by JE DNA vaccine was also compared, our research showed the percentage of CD4 ~ T cells in spleen cells and anti- gen-specific CTL activities in the pJME/ICAM-1 and pJME ＋ pICAM-1 vaccinated group were significantly higher than that in the pJME and JE inactivated vaccine group（ P 〈 0.05 ）. Conclusion： ICAM-1 coding gene can promote the maturation of spleen DC induced by JE DNA vaccine, and amplify costimulatory signals that be independent of B7 molecules.