cGAS在介导pUC18-CpG免疫增强效果中的作用和机制探讨

In recent studies, cGAS was found to be a novel cytosolic DNA sensor that activates innate immunity, and controls the progress of adaptive immunity. Our previous studies have shown that pUC18-CpG could greatly enhance the efficacy of a swine FMD killed vaccine，and a live attenuated PRRS vaccine，respectively. However, besides the membrane-related receptor TLR9 that recognizes CpG DNA, whether or not cGAS mediates the adjuvanticity of pUC18-CpG is still not clear. In this study, an assumption is made that cGAS also recognizes pUC18-CpG and mediates its adjuvanticity. Hence, the gene coding for cGAS is cloned, and the protein expressed and purified. DNA-binding experiment is carried out by use of the Biotin-Avidin System. The bioactivity of cGAS is tested by western blotting. HEK293T-STING cell line is prepared. The cGAS-related signaling pathways that mediate the adjuvanticity of pUC18-CpG are studied via western blotting，RNAi, EMSA, etc. RT-qPCR is used for the quantification of TypeⅠIFN mRNAs. RNA-Seq is carried out to analyze the differences in the expression profiles of TypeⅠIFN―regulated genes among different swine PBMCs groups. By analyzing the results, the role of cGAS and its related mechanisms in mediating the adjuvanticity of pUC18-CpG are discussed.

cGAS是近期报道的一种在激活先天免疫、调节适应性免疫中起重要作用的DNA胞质受体。前期研究发现，pUC18-CpG可显著提高猪口蹄疫灭活疫苗、蓝耳病活疫苗的免疫保护效果；然而，除CpG DNA膜受体TLR9外，pUC18-CpG是否通过cGAS发挥其佐剂效应尚未有报道。本研究假设cGAS参与介导pUC18-CpG的免疫增强作用，由此克隆猪cGAS基因，表达、纯化cGAS蛋白；以生物素-亲和素系统验证cGAS体外结合DNA的能力，免疫印迹法间接检测其生物学活性；构建HEK293T-STING细胞系，利用ELISA、RNAi等方法研究pUC18-CpG是否激活与cGAS相关的信号通路，定量PCR检测细胞中Ⅰ型干扰素mRNA转录水平；利用RNA-Seq分析猪PBMCs中cGAS介导的Ⅰ型干扰素相关基因转录情况。综合分析研究结果，初步阐明cGAS在pUC18-CpG免疫增强效果中的作用和机制。

环鸟苷酸-腺苷酸合成酶是一种普遍存在于细胞质中的DNA模式识别受体，在激活先天免疫、调节适应性免疫中起重要作用。本研究首先扩增了猪cGAS、STING、TBK1和IRF3基因序列，将其定向克隆入真核表达载体。在细胞水平，利用pUC18-CpG、人工合成CpG ODN以及pCpG free质粒分别刺激RAW264.7细胞，结果表明三者均能够上调I型干扰素和促炎性因子表达。通过使用CpG ODN拮抗剂、cGAS-STING-TBK1信号通路抑制剂BX795，研究TLR9、cGAS在介导pUC18-CpG固有免疫识别中的作用。结果显示，使用BX795能够有效抑制pUC18-CpG刺激细胞产生I型干扰素水平，而拮抗剂并未干扰pUC18-CpG刺激细胞产生I型干扰素，提示pUC18-CpG主要通过cGAS-STING-TBK1信号通路发挥作用。利用双荧光素酶报告基因系统，在细胞水平验证了cGAS、STING共转染对IFN-β启动子、NF-κB途径的激活作用，同时初步研究病毒免疫逃逸蛋白对cGAS-STING-TBK1通路的抑制作用。.验证了pUC18-CpG、人工合成CpG ODN对Eg95、口蹄疫灭活抗原以及gp90抗原的免疫增强作用。以CpG ODN和pUC18-CpG作为佐剂配伍EG95免疫小鼠后均能显著提高抗体水平，上调Th1型细胞因子的表达。将CpG ODN、pUC18-CpG分别同口蹄疫O型抗原混合，每周分离血清，检测抗体效价。结果表明，合成CpG ODN和pUC18-CpG作为佐剂在均能使免疫小鼠提前产生抗原特异性抗体, 并显著提高首免后的抗体水平。.将pUC18-CpG与口蹄疫灭活疫苗配伍免疫12月龄奶牛，结果表明pUC18-CpG作为佐剂能够显著提高疫苗免疫后的抗体水平。以pCpG free、CpG ODN以及PBS分别刺激牛外周血单个核细胞，提取细胞RNA进行转录组测序分析，结果表明pCpG free、CpG ODN刺激后能上调表达多种免疫调节基因。对两者刺激产生的差异表达基因进行GO分析和KEGG分析表明，CpG ODN刺激产生的细胞因子具有更高的广谱性，而pCpG free刺激能够更好的模拟宿主胞浆模式识别受体对病毒DNA的识别机制。研究结果对于探索外源DNA的免疫识别机制以及新型核酸佐剂的开发具有一定的指导意义。