IgG Fc基因增强乙型脑炎DNA疫苗细胞免疫效应的分析

Cellular immune effect of DNA vaccine derived from Japanese encephalitis virus enhanced by immunoglobulin G Fc fragment

目的评价IgG Fc段编码基因不同构建方式对乙型脑炎(JE)DNA疫苗细胞免疫应答的影响。方法以套式RT-PCR法获取BALB/c鼠IgG Fc编码基因,构建含乙型脑炎病毒(JEV)pr ME蛋白与IgG Fc编码基因以及单纯IgG Fc编码基因重组质粒,分别命名pJME/IgG Fc和pIgG Fc,以不同免疫原肌注免疫BALB/c小鼠,经流式细胞仪检测不同免疫原免疫鼠后脾T淋巴细胞亚群及Th细胞内细胞因子(IFN-γ、IL-4)变化,乳酸脱氢酶(Lactate dehydrogenase,LDH)法测定细胞毒性T淋巴细胞(Cytotoxic T lymphocytes,CTL)活性。数据进行单因素方差分析。结果 pJME/IgG Fc组CD4+T淋巴细胞比例为(66.64±5.84)%,明显高于pJME+pIgG Fc、pJME、JE灭活疫苗和pc DNA3.1(+)组(P<0.05),pJME+pIgG Fc组CD4+T淋巴细胞比例(60.55±2.09)%,高于pJME组(52.86±1.92)%,也高于JE灭活疫苗组(55.77±1.62)%(P<0.05),pc DNA3.1(+)组CD4+T淋巴细胞比例为(37.82±4.93)%,明显低于其他组(P<0.05);pJME/IgG Fc与pJME+pIgG Fc免疫组CD8+T细胞比例较空载体pc DNA3.1(+)及灭活疫苗组升高(P<0.05)。pJME/IgG Fc免疫组CTL活性为(46.92±1.97)%,明显高于其它组(P<0.05)。pJME/IgG Fc、pJME+pIgG Fc免疫组IFN-γ+CD4+T淋巴细胞比例分别为(37.90±4.79)%、(21.53±4.61)%,明显高于其它组(P<0.05)。结论相对于pJME+pIgG Fc联合免疫组,pJME/IgG Fc融合质粒免疫组能够诱导更强的细胞免疫反应。

Objective To investigate the specific cellular immune effect induced by plasmid DNA vaccine derived fromJapanese encephalitis virus（JEV）with various forms of IgG Fc gene. Methods Gene encoding IgG Fc was amplified bynested-RT-PCR technique from BALB/c murine spleen cells. Genetic fusion of pr ME protein and IgG Fc gene,and pure IgG Fc gene were subcloned respectively,named as pJME/IgG Fc and pIgG Fc . For the immunization of the BALB/c mice,the Tlymphocyte subsets and the levels of Th cells intracellular cytokine IFN-γ and IL-4 in the splenic cells suspension of the micewere evaluated by flow cytometric analysis. The CTLs activity were assessed by lactic dehydrogenase（LDH）release method.The data were analyzed by single factor analysis of variance. Results The percentage of CD4＋T cells in the pJME/IgG Fc vaccinated groupwas（66.64±5.84）%,significantly higher than other groups（P〈0.05）. The percentage of CD4＋T cells in thepJME＋pIgG Fc vaccinated groups were（60.55±2.09）%,higher than that in the pJME（52.86±1.92）% and JE inactivatedvaccine（55.77±1.62）%vaccinated group（P〈0.05）. The percentage of CD4＋T cells in the pc DNA3.1（＋）groupwas（37.82±4.93）%,lower than other groups（P〈0.05）. The percentage of CD8＋T cells in the pJME/IgG Fc and pJME＋pIgG Fc vaccinatedgroups were higher than that in the empty plasmid and JE inactivated vaccine vaccinated groups（P〈0.05）. The CTL activitiesinduced by pJME/IgG Fc vaccinated groupwere（46.92±1.97）%,higher than other groups（P〈0.05）. The percentage of IFN-γ＋CD4＋T cells from the pJME/IgG Fc , pJME ＋ pIgG Fc vaccinated groups were（37.90 ± 4.79）% and（21.53 ± 4.61）%respectively, significantly higher than that in other groups（P〈0.05）. Conclusion Compared with pJME＋pIgG Fc combinedimmunization group,pJME/IgG Fc fusion plasmid immunization groupwould induce much more intense cellular immuneresponse.