中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2009年
5期
257-261
,共5页
翟永贞%李喜梅%周言%张丹%邓宝成%冯国和
翟永貞%李喜梅%週言%張丹%鄧寶成%馮國和
적영정%리희매%주언%장단%산보성%풍국화
脑炎病毒,日本%疫苗,DNA%粒细胞巨噬细胞集落刺激因子%免疫,细胞
腦炎病毒,日本%疫苗,DNA%粒細胞巨噬細胞集落刺激因子%免疫,細胞
뇌염병독,일본%역묘,DNA%립세포거서세포집락자격인자%면역,세포
Encephalitis virus,Japanese%Vaccines,DNA%Granulocyte-macrophage colony-stimulating factor%Immunity,cellular
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入中华仓鼠卵巢细胞(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3 d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3 d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3 d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JE DNA疫苗的细胞免疫应答效应.
目的 研究粒細胞-巨噬細胞集落刺激因子(GM-CSF)編碼基因對乙型腦炎(JE)DNA疫苗細胞免疫應答的影響.方法 套式RT-PCR法穫取BALB/c小鼠GM-CSF編碼基因,構建含乙型腦炎病毒(JEV)前膜(prM)-包膜(E)蛋白與GM-CSF編碼基因以及單純GM-CSF編碼基因的重組質粒,分彆命名為pJME/GM-CSF和pGM-CSF.脂質體法轉染上述質粒入中華倉鼠卵巢細胞(CHO),免疫熒光法檢測編碼蛋白的錶達與分佈.流式細胞儀檢測經不同組閤的免疫原免疫後小鼠脾T淋巴細胞亞群及Th細胞內細胞因子(IFN-γ、IL-4)變化,LDH法測定CTL活性.數據行單因素方差分析及最小顯著差數法比較.結果 重組質粒pJME/GM-CSF與pGM-CSF經鑒定構建正確,所編碼的蛋白主要分佈于胞質,少量分佈于胞膜.pJME/GM-CSF組CD4+T淋巴細胞比例為(33.90±0.79)%,明顯高于其他組(t值分彆為9.818、6.804、6.594、10.061、9.380和17.675,均P<0.05).pJME+pGM-CSF同時註射組及pGM-CSF註射3 d後接種pJME組CD4+T淋巴細胞比例分彆為(29.83±0.61)%、(29.70±0.51)%,高于pJME註射3 d後接種pGM-CSF組的(27.69±0.50)%(t=3.466、t=3.255,P<0.05).pJME/GM-CSF組與pJME+pGM-CSF同時註射組CD8+T淋巴細胞比例較空載體(pcDNA3.1+)組及JE滅活疫苗組升高(t值分彆為3.811、2.627、10.537和3.811,均P<0.05).pJME/GM-CSF組CTL活性為(51.48±0.10)%,明顯高于其他組(t值分彆為22.868、13.823、5.377、32.287、34.632和53.795,均P<0.05).pJME/GM-CSF組、pJME+pGM-CSF同時註射組及pGM-CSF註射3 d後接種pJME組IFN-γ/IL-4比值分彆為19.13±1.36、12.32±0.82、7.05±0.43,明顯高于其他組(P<0.05).結論 GM-CSF編碼基因可增彊JE DNA疫苗的細胞免疫應答效應.
목적 연구립세포-거서세포집락자격인자(GM-CSF)편마기인대을형뇌염(JE)DNA역묘세포면역응답적영향.방법 투식RT-PCR법획취BALB/c소서GM-CSF편마기인,구건함을형뇌염병독(JEV)전막(prM)-포막(E)단백여GM-CSF편마기인이급단순GM-CSF편마기인적중조질립,분별명명위pJME/GM-CSF화pGM-CSF.지질체법전염상술질립입중화창서란소세포(CHO),면역형광법검측편마단백적표체여분포.류식세포의검측경불동조합적면역원면역후소서비T림파세포아군급Th세포내세포인자(IFN-γ、IL-4)변화,LDH법측정CTL활성.수거행단인소방차분석급최소현저차수법비교.결과 중조질립pJME/GM-CSF여pGM-CSF경감정구건정학,소편마적단백주요분포우포질,소량분포우포막.pJME/GM-CSF조CD4+T림파세포비례위(33.90±0.79)%,명현고우기타조(t치분별위9.818、6.804、6.594、10.061、9.380화17.675,균P<0.05).pJME+pGM-CSF동시주사조급pGM-CSF주사3 d후접충pJME조CD4+T림파세포비례분별위(29.83±0.61)%、(29.70±0.51)%,고우pJME주사3 d후접충pGM-CSF조적(27.69±0.50)%(t=3.466、t=3.255,P<0.05).pJME/GM-CSF조여pJME+pGM-CSF동시주사조CD8+T림파세포비례교공재체(pcDNA3.1+)조급JE멸활역묘조승고(t치분별위3.811、2.627、10.537화3.811,균P<0.05).pJME/GM-CSF조CTL활성위(51.48±0.10)%,명현고우기타조(t치분별위22.868、13.823、5.377、32.287、34.632화53.795,균P<0.05).pJME/GM-CSF조、pJME+pGM-CSF동시주사조급pGM-CSF주사3 d후접충pJME조IFN-γ/IL-4비치분별위19.13±1.36、12.32±0.82、7.05±0.43,명현고우기타조(P<0.05).결론 GM-CSF편마기인가증강JE DNA역묘적세포면역응답효응.
Objective To study the adjuvant effect of granulocyte-macrophage colony-stimulating 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/GM-CSF and pJME+pGM-CSF vaccinated groups were both higher than that in empty vector (pcDNA 3.1+) group and JE inactivated vaccine vaccinated group (t values were 3.811, 2.627, 10.537, and 3.811, all P<0.05). The CTL activity in pJME/GM-CSF vaccinated group was (51.48±0.10)%, which was higher than those in other groups (t values were 22.868, 13.823, 5.377, 32.287, 34.632, and 53.795, all P<0.05). The IFN-γ/IL-4 ratios in pJME/GM-CSF, pJME+pGM-CSF (0) and pJME + pGM-CSF (-3) vaccinated groups were (19.13±1.36), (12.32±0.82) and (7.05±0.43), respectively, which were higher than those in other groups (P<0.05). Conclusion GM-CSF coding gene could enhance the cellular immune response induced by Japanese encephalitis DNA vaccine.
