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猪白细胞介素2与融合白细胞介素4 6基因共表达的免疫效应研究
Study on Immune Synergistic Effect of Co-expression of Pig Fusion Interleukin-4/6 and Interleukin-2 Gene
杨璐一#,肖永乐#,万小平,罗公明,王晔,陈义辉,孙文魁,高荣*
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作者单位: (四川大学生命科学学院,生物资源与生态环境教育部重点实验室,动物疾病防控和食品安全四川省重点实验室,成都 610064)
中文关键字:猪融合白细胞介素4/6基因;猪白细胞介素2基因;共表达;免疫;小鼠
英文关键字:pig fusion interleukin-4/6 gene; interleukin-2 gene; co-expression; immunity; mouse
中文摘要:目的  构建猪融合白细胞介素4/6与猪白细胞介素2基因的共表达载体,研究其共表达对小鼠免疫的协同效应。方法  2A自剪接技术首次构建猪融合白细胞介素4/6与白细胞介素2基因的共表达重组质粒,以壳聚糖纳米材料包裹制成纳米颗粒,进行体外转染HEK293细胞,提取总RNA 进行RT-PCR分析,最后肌肉注射小鼠进行体内实验并分析。结果  成功构建了重组共表达质粒VRIL4/6-2;壳聚糖包裹后转染HEK293细胞,48 h后收集细胞,RT-PCR检测显示目的基因能够在HEK293细胞中高效地转录与表达。小鼠接种实验结果表明,实验组血清中的IgGIgG2a的含量比对照组显著增加,并且VRIL4/6-2-CS接种组的小鼠体液免疫水平明显增高(P0.05)VRIL4/6-2实验组的CD4+淋巴细胞显著多于其它实验组(P0.05);实验组IL-2IL-4IL-6IL-23基因的表达水平明显高于对照组(P0.05),实验组小鼠外周血白细胞、血小板和血红蛋白的含量均显著高于对照组(P<0.05)结论  VRIL4/6-2共表达质粒能够更好的增强动物体液免疫和细胞免疫机能,可作为提高动物体液免疫和细胞免疫的经济高效安全新型免疫调节剂,具有潜在的重要应用前景。
英文摘要:Objective  To construct the co-expression recombinant vector with pig fusion interleukin-4/6 and interleukin-2 genes and its synergistic effect on immunity of mice. Methods  The co-expression recombinant plasmid with pig fusion interleukin-4/6 and interleukin-2 genes was firstly constructed using self cleave 2Atechnique, and then it was wrapped with chitosan to transfect HEK293 cells and cultured for 48 h. The total RNA was extracted from transfected cells for RT-PCR analysis. Finally, the recombinant plasmid nanoparticles were intramuscularly injected into KM mice to evaluate the immune synergistic effect. Results  The co-expression recombinant plasmid with pig fusion interleukin-4/6 and interleukin-2 genes was successfully constructed by self-cleavage2Atechnique and named as VRIL4/6-2. After transfection, RT-PCR assay showed that the recombinant interleukin genes were efficiently expressed in transfected HEK293 cells. The results showed that the production of IgG and IgG2awas significantly raised in injected mice, and the VRIL4/6-2 group showed the highest humoral immunity elevation than the other two injected mice (P<0.05). The number of CD4+ lymphocytes of VRIL4/6-2 treated group was markedly higher than that of the other two injected groups (P<0.05), and the expression levels of IL-2, IL-4, IL-6 and IL-23 genes of the treated groups  significantly elevated in comparison with those of control group (P<0.05). The numbers of leukocytes and platelets as well as hemoglobin levels were significantly increased in the injected mice (P<0.05). Conclusion  VRIL4/6-2 performs significant synergistic effect to enhance host immunity which can be further utilized to develop safe and effective immunoadjuvant for the control and prevention of animal disease.
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