摘要
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。
AIM： To construct a novel Ag85B DNA vaccine based on the genetic adjuvant of T-bet known as Th1 transcription factor and to research the immunoregulation function of the DNA complex vaccine. METHODS： Ag85B gene and T-bet gene were amplified by RT-PCR, and cloned into pcDNA3. 1 plasmid to construct recombinant plasmids pcDNA3-Ag85B and pcDNA3-T-bet, respectively. The recombinant plasmids were transfected into RAW264. ？ cells using LipofectamineTM 2000 reagent to detect the expressions of Ag85B and T-bet proteins by Western blotting. BALB/c mice were immunized by three intramuscular inoculations with pcDNA3, 1-FLAG-T-bet in combination with pcDNA3.1-FLAG-Ag85B. Two weeks after the last immunization, the anti-Ag85B antibody titres in sera were tested by ELISA. Meanwhile, spleen lymphocyte suspension was cultured in the context of Ag85B, and then the secretion of cytokines in the culture fluid was tested by ELISA. RESULTS： Plasmid proteins were successfully expressed in a dose-dependent manner. Additionally, T-bet/Ag85B complex not only induced obviously higher IgG2a titre with the lower IgG1, but also stimulated the increased secretion of IFN-y and IL-2 with the concomitant repression of IL-4 and IL-10. CONCLUSION： T-bet can enhance Ag85B-specific IgG2a antibody response and convert T cell subsets to a Th1-predominant immune response.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第7期680-683,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81041083
81172778)
安徽省自然科学基金(1208085QH162)
