论文部分内容阅读
目的:构建以Th1转录因子T-bet为基因佐剂的Ag85B新型DNA疫苗,并研究其免疫调控作用。方法:RT-PCR法扩增出Ag85B基因和T-bet基因,克隆入pcDNA3.1质粒构建T-bet和Ag85B真核表达质粒,脂质体法转染重组质粒至RAW264.7细胞系,Western blot法检测质粒蛋白表达情况。3次肌肉注射免疫BALB/c小鼠,末次免疫2周后,ELISA法检测血清中抗Ag85B抗体滴度。同时将脾脏淋巴细胞悬液于Ag85B刺激下培养,ELISA法检测培养液中细胞因子分泌情况。结果:质粒蛋白成功表达,并且质粒剂量与质粒蛋白表达水平呈正相关。此外,T-bet/Ag85B不仅诱导IgG2a滴度显著增高伴随IgG1显著降低,而且还刺激IL-2/IFN-γ(Th1类)分泌增加伴随IL-4/IL-10(Th2类)减少。结论:T-bet增强抗Ag85B特异性IgG2a抗体反应,并诱导显著的Th1细胞优势免疫。
OBJECTIVE: To construct a novel Ag85B DNA vaccine adjuvanted with Th1 transcription factor T-bet, and study its immunomodulatory effects. Methods: Ag85B gene and T-bet gene were amplified by RT-PCR, cloned into pcDNA3.1 plasmid to construct T-bet and Ag85B eukaryotic expression plasmids, and then transfected into RAW264.7 cell line by lipofectamine blot method to detect the expression of plasmid protein. BALB / c mice were immunized with 3 intramuscular injections. Serum anti-Ag85B antibody titers were measured by ELISA after two weeks of last immunization. At the same time, the spleen lymphocyte suspension was cultured under Ag85B stimulation, and the secretion of cytokines in the culture fluid was detected by ELISA. Results: Plasmid protein was successfully expressed, and the plasmid dose was positively correlated with the expression of plasmid protein. In addition, T-bet / Ag85B not only induced a significant increase of IgG2a titers with a significant decrease of IgG1, but also stimulated an increase of IL-2 / IFN-γ (Th1) secretion accompanied by a decrease of IL-4 / IL-10 (Th2). CONCLUSION: T-bet enhances the anti-Ag85B-specific IgG2a antibody response and induces significant Th1 cellular predominance.