【摘要】 cGAS作为一种新型的胞质DNA受体，在宿主抵抗DNA病毒而触发的天然免疫中起着至关重要的作用。血清4型禽腺病毒(Fowl adenovirus serotype 4,FAdV-4)是无囊膜的一种双链DNA病毒，可引起鸡肝炎-心包积液综合征。为明确鸡cGAS(chcGAS)基因功能，探究在鸡肝癌(LMH)细胞中过表达chcGAS以及FAdV-4感染前后细胞定位的变化情况。本研究针对chcGAS序列设计引物进行PCR扩增，并分析该基因序列与其他物种之间的同源性以及预测该基因的结构域，构建重组质粒pET-32a-chcGAS进行原核表达，通过SDS-PAGE和Western blot鉴定，利用激光共聚焦观察FAdV-4感染LMH细胞前后chcGAS细胞定位变化。结果表明，本试验克隆得到大小为1 317 bp的chcGAS基因，与其他物种同源性为50.5%～84.4%,SDS-PAGE分析结果显示，chcGAS蛋白主要以可溶性蛋白质的形式在上清液处表达，目的蛋白质相对分子质量为75 000,与预期大小相符。亚细胞定位结果显示，FAdV-4感染可导致定位于细胞核膜上的chcGAS蛋白转移到细胞质中。本研究结果为进一步研究FAdV-4与cGAS-STING信号通路的关联调控机制提供了科学依据。更多还原

【Abstract】 cGAS plays a key role in the host’s innate immune response against DNA viruses as a receptor for DNA recognition within the cytoplasm. Fowl adenovirus serotype 4(FAdV-4) is a double-stranded DNA virus without an envelope, and can cause hepatitis-pericardial effusion syndrome in chicken. In order to clarify the function of chicken cGAS(chcGAS) gene, the overexpression of chcGAS in chicken liver cancer(LMH) cells and the changes of cell location before and after FAdV-4 infection were investigated. In this study, the primers were designed according to the chcGAS sequence, and the chcGAS gene was amplified by PCR. The homology between the gene sequence and other species was analyzed, and the domain was predicted. The recombinant plasmid pET-32a-chcGAS was constructed for prokaryotic expression, identified by SDS-PAGE and Western blot, and the localization changes of chcGAS in LMH cells before and after FAdV-4 infection were observed by laser confocal microscopy. The results showed that the chcGAS gene with a size of 1 317 bp was successfully cloned, and the homology with other species was 50.5%-84.4%. The results of SDS-PAGE analysis indicated that chcGAS protein was mainly expressed in the supernatant in the form of soluble protein. The relative molecular mass of target protein was 75 000, which was consistent with the expected size. The results of subcellular localization showed that FAdV-4 infection could lead to transfer of chcGAS protein localized on the nuclear membrane into the cytoplasm. These results can provide a scientific basis for further studying the correlation regulatory mechanism between FAdV-4 and cGAS-STING signaling pathways.更多还原