鸭视黄酸诱导基因蛋白I(RIG-I)抗禽流感病毒的分子调控机理

视黄酸诱导基因蛋白I(RIG-I)是一类重要的病毒感受分子，能广泛抑制RNA和DNA病毒的增殖。申请人发现高致病性流感病毒感染引起鸭RIG-I基因的持续性高表达，诱导干扰素和炎症因子表达的持续性上调，进而导致机体的过度炎症反应。然而，鸭应答流感病毒感染免疫反应中,RIG-I基因的分子调控机理尚不清楚。在构建鸭RIG-I基因共表达网络和筛选RIG-I互作lncRNA的基础上，申请人拟利用DNA亲和纯化质谱筛选鸭RIG-I基因的转录因子；检测转录因子过表达/敲除DEF细胞系多种病毒生长曲线、RIG-I基因的mRNA和蛋白表达量、基因表达图谱；验证转录因子与RIG-I启动子及蛋白互作；鉴定转录因子和RIG-I互作的lncRNA，并解析互作lncRNA的作用模型。该研究目标的实现可鉴定RIG-I的转录因子、验证其功能结构域，解析鸭RIG-I基因抗流感病毒的分子调控机理，以助力鸭抗病优良品系的培育。

Retinoic acid-induced gene I is a critical viral sensor distributing in cytoplasm. After recognizing different signaling molecules (i.e. dsRNA), RIG-I induces expression of interferon and inflammatory factors to inhibit replication of a broad range of RNA and DNA viruses. The applicant has found that: 1. the highly pathogenic H5N1 (A/duck/Hubei/49/05, DK/49) virus-infected ducks had continuously high expression of RIG-I and inflammatory factors, resulting in excessive inflammation and even death; 2. construction the co-expressional network of RIG-I using 21 transcriptomes of ducks infected with a highly (DK/49) or weakly (A/goose/Hubei/65/05, GS/65) H5N1 virus; 3. identification of three candidate lncRNAs co-expressing with RIG-I in the above 21 duck transcriptomes through the WGCNA (weighted gene co-expression network) analysis. However, the mechanism about how the RIG-I is regulated during immune response to viral infection is largely unclear. In this project, we will perform the following works to identify transcript factor and interacted lncRNA of duck RIG-I, define their functional domain and network: 1. identify transcript factor of RIG-I using the DNA affinity mass spectrometry analysis; 2. creating duck embryonic fibroblasts (DEF) cells, which are over-expressed or deficient transcript factor and its truncations of duck RIG-I using gene-editing technology, such as piggyBac transposon-mediated gene transfer system or CRISPR/CAS9 system; 3. calculate viral growth curve of several viruses (i.e. influenza A viruses, new castle virus and duck hepatitis virus), qualify mRNA and protein expression of RIG-I and construct gene expressional profiles of the above DEF cells before and after infected by viruses; 4. confirm interaction between transcript factor and RIG-I protein through molecular biological experiments, such as co-immunoprecipitation and yeast two-hybrid analysis; 6. identify lncRNA binding to transcript factor and RIG-I protein using crosslinking-immunoprecipitation followed by sequencing (CLIP-seq); 7. test regulation of lncRNA to expression of RIG-I and confirm interaction between lncRNA and RIG-I/transcript factor in DEF cells. These efforts will provide new insights in the molecular regulator model for duck RIG-I gene in immune response to avian influenza infections and it will be informative to the breeding of duck lines showing resistance to disease, such as avian influenza.