鸡三种天然免疫相关受体和配体荧光定量PCR检测方法的建立及应用

Development and Application of Quantitative PCR Assay for Detection of Three Innate Immunity-related Cellular Receptors and Ligands in Chickens

为建立检测鸡天然免疫相关受体(Ch-MDA5、Ch-TLR3和Ch-TLR7)及其相应配体(MAVS、TRIF和MyD88)的荧光定量PCR方法并评价其应用效果,设计和筛选特异性PCR引物,制备各基因的质粒标准品,绘制荧光定量PCR的标准曲线,并对方法的灵敏度、重复性和特异性进行检验。结果表明,所建立的检测方法敏感度高、特异性强、检测线性范围广、重复性好。应用建立的方法检测新城疫油乳剂灭活苗免疫SPF鸡后上述基因表达的变化,发现免疫后6h各基因的表达水平达到峰值,表明疫苗免疫迅速有效地激活了机体的天然免疫应答。建立了与鸡抗病毒感染天然免疫相关的3种细胞受体和相应配体的定量检测方法,可应用于病毒致病机制、鸡用新型疫苗、免疫增强剂以及抗病毒药物的研究与开发。

The main purpose of this study is to establish quantitative PCR （qPCR） for the detection of three pattern recognition receptors （chicken Toll-like receptor 3,Toll-like receptor 7 ,and melanoma differentia- tion-associated protein 5） and their respective ligands （TRIF,MyD88 and MAVS） in chickens. We designed and screened PCR primers for the 6 genes. Specific PCR products of the genes were utilized to construct re- combinant plasmids,which served as template standards to establish standard curves for the assays. The as- says developed in this study were sensitive, specific and could stably detect related genes of wide concentra- tion ranges. Further, the assays were applied in the analysis of related gene expression in specific-pathogen- free （SPF） chickens after immunization with oil-emulsion vaccine against Newcastle disease virus. The up- regulations of expression of all 6 genes were peeked at six hours post vaccination, and at the same time,the mRNA level of IFN-13 increased to 252 times. This indicated the efficacy of innate immunity signaling after vaccination. In conclusion, the qPCR developed in this study may be applied in viral pathogenesis research, and novel vaccine,immnopotentiator and anti-viral drug development.