摘要
目的:探讨汉坦病毒H8205株G1-G2/人源性白细胞介素2(IL-2)融合基因在非洲绿猴肾(Vero)细胞获得稳定表达的可能性,评价融合基因pcDNA3.1/HisB-IL-2-G1-G2的DNA免疫效果。方法:在脂质体介导下,将pcDNA3.1/HisB-IL-2-G1-G2导入Vero细胞,通过G418筛选获得阳性克隆,用间接免疫荧光(IFAT)、十二烷基磺酸钠-聚丙烯酰胺电泳(SDS-PAGE)等方法检测其在Vero细胞中稳定表达的情况。采用肌肉注射质粒的方法来免疫Balb/c小鼠,免疫3次,加强2次,间隔2周,然后用酶联免疫吸附法(ELISA)检测血清特异性抗体,用空斑减少中和试验检测中和抗体。结果:转染了pcDNA3.1/HisB-IL-2-G1-G2后在Vero细胞培养上清和胞质中有融合蛋白的表达,其相对分子量为110 ku。pcDNA3.1/HisB-IL-2-G1-G2可诱导小鼠产生特异性抗汉坦病毒抗体和中和抗体,其效价均高于其他对照组,差异有显著性(P<0.05)。结论:融合基因在脂质体介导下能导入Vero细胞并获得稳定表达。融合基因真核载体疫苗免疫Balb/c小鼠可诱导特异性体液免疫应答,为进一步将其用于我国汉坦病毒疫苗研制提供了依据。
Objective To explore the feasibility of stable expression of Hantavirus G1-G2/human interleukin-2（IL-2） fusion gene in Veto cells, and evaluate its immuno-protection effects on Balb/c mice. Methods Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1-G2 plasmid by lipofectamine, the positive cells were selected by G418. Immunofluorescent antibody test（IFAT） and sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） were used to determine the stability of transfection and the expression of recombinant protein. Each mouse was inoculated 3 times via intramuscular injection, 2 boosts were administrated at 2-week intervals. Serum anti-Hantavirus antibodies were detected by enzyme linked immunosorbent assay （ELISA）, and neutralizing antibodies （NAb） were detected by plaque reduction neutralization test. Results The molecular weight of fusion protein expressed in Vero cells was 110 ku, corresponding to that of the estimated size. pcDNA3.1/HisB-IL-2-G1-G2 elicited significant antibody responses and induced neutralizing antibodies production. The titer of antibodies induced by pcDNA3.1/HisB-IL-2-G1-G2 was much higher than that in other groups（P〈0.05） Conclusions IL-2/G1-G2 fusion gene could be transferred in Vero cells and stably expressed. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing Hantavirus G1-G2/IL-2 gene. This study lays the foundation for further development of therapeutic Hantavirus vaccine.
出处
《内科理论与实践》
2007年第4期256-259,共4页
Journal of Internal Medicine Concepts & Practice
基金
国家自然科学基金资助项目(项目编号:30170819)
