中文摘要：

目的研究粒细胞-巨噬细胞集落刺激因子（GM—CSF）编码基因对乙型脑炎（JE）DNA疫苗细胞免疫应答的影响。方法套式RT—PCR法获取BALB／c小鼠GM—CSF编码基因，构建含乙型脑炎病毒（JEV）前膜（prM）-包膜（E）蛋白与GM-CSF编码基因以及单纯GM—CSF编码基因的重组质粒，分别命名为pJME／GM—CSF和pGM—CSF。脂质体法转染上述质粒人中华仓鼠卵巢细胞（CHO），免疫荧光法检测编码蛋白的表达与分布。流式细胞仪检测经不同组合的免疫原免疫后小鼠脾T淋巴细胞亚群及Th细胞内细胞因子（IFN-γ、IL-4）变化，LDH法测定CTL活性。数据行单因素方差分析及最小显著差数法比较。结果重组质粒pJME／GM—CSF与pGM—CSF经鉴定构建正确，所编码的蛋白主要分布于胞质，少量分布于胞膜。pJME／GM—CSF组CD4^＋T淋巴细胞比例为（33．90±0．79）％，明显高于其他组（t值分别为9．818、6．804、6．594、10．061、9．380和17．675，均P％0．05）。pJME＋pGM—CSF同时注射组及pGM—CSF注射3d后接种pJME组CD4^＋T淋巴细胞比例分别为（29．83±0．61）％、（29．70±0．51）％，高于pJME注射3d后接种pGM—CSF组的（27．69±0．50）％（t=3．466、t=3．255，P〈0．05）。pJME／GM—CSF组与pJME＋pGM—CSF同时注射组CD8^＋T淋巴细胞比例较空载体（pcDNA3．1＋）组及JE灭活疫苗组升高（t值分别为3．811、2．627、10．537和3．811，均P〈0．05）。pJME／GM—CSF组CTL活性为（51．48±0．10）％，明显高于其他组（t值分别为22．868、13．823、5．377、32．287、34．632和53．795，均P〈0．05）。pJME／GM—CSF组、pJME＋pGM—CSF同时注射组及pGM—CSF注射3d后接种pJME组IFN-γ／IL-4比值分别为19．13±1．36、12．32±0．82、7．05±0．43，明显高于其他组（P〈0．05）。结论GM—CSF编码基因可增强JEDNA疫苗的细胞免疫应答效应。

英文摘要：

Objective To study the adjuvant effect of granulocyte-macrophage colonystimulating factor （GM-CSF） coding gene on cellular immunity induced by Japanese encephalitis （JE） virus DNA vaccine. Methods GM-CSF coding gene was amplified by nested-reverse transcriptase- polymerase chain reaction （RT-PCR） technique from BALB/c murine spleen cells. Recombinant plasmids pJME/GM-CSF and pGM-CSF were constructed by JE virus （JEV） prM-E protein with GM-CSF coding gene or GM-CSF coding gene only, respectively. The plasmids were transfected into China hamster ovary （CHO） cells by Lipofectamine 2000. The coding protein expressions and distributions were detected by immunofluorescence. The BALB/c mice were vaccinated with indicated immunogens with or without GM-CSF gene. The changes of T lymphocyte subsets in the spleen and levels of intracellular cytokines, such as interferon （IFN）-γ and interleukin （IL）-4 of splenic cells from mice immunized with different immunogens were evaluated by flow cytometry. The cytotoxicity T lymphocyte （CTL） activity was assessed by lactate dehydrogenase （LDH）. The data were compared by one-factor analysis of variance and least significant difference. Results The constructed recombinant pGM-CSF and pJME/GM CSF were confirmed by restrict enzyme digestion and DNA sequencing. The expressions of the above proteins were mainly in the cytoplasm and minor on cell membrane. The percentage of CD4^＋ T lymphocytes in pJME/GM-CSF vaccinated group was （33.90±0.79） %, which was significantly higher than that of in other groups （t values were 9. 818, 6. 804, 6. 594, 10. 061, 9. 380, and 17. 675, all P〈0.05）. The percentages of CD4^＋ T lymphocytes in pJME ＋pGM-CSF （0） and pJME＋pGM-CSF （-3） vaccinated groups were （29. 83±0. 61）% and （29.70±0.51）%, respectively, which were both higher than that in pJME＋ pGM CSF （＋3） vaccinated group of （27. 69±0. 50）% （t=3. 466, t=3. 255, both P〈0. 05）. The percentages of CD8＋ T cells in pJME