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为建立山羊IL-2基因SYBR GREENⅠ实时荧光定量RT-PCR检测方法,根据Gen Bank中山羊IL-2基因(登录号:KT934548),设计1对特异性引物,用于扩增目的基因,并将目的基因克隆于p MD-19-T载体,转化至大肠杆菌DH5α,经质粒PCR及序列测定鉴定后获得阳性重组质粒,作为标准品模板建立SYBR GREENⅠRTFQ-PCR标准曲线和溶解曲线,进行特异性、重复性和敏感性试验。并应用所建立的方法,检测Con A刺激健康山羊PBMC后0 h、2 h、4 h、6 h、8 h、10 h、12 h、24 h和48 h不同时间点IL-2基因转录的动态变化。结果表明,当质粒标准品稀释度在7.2×10~9~7.2×10~5 copies/μL扩增曲线的Ct值与浓度间具有良好的线性关系,相关系数为-0.996;熔解曲线为特异性单峰,组内变异系数为0.306%~1.458%,组间变异系数为0.514%~1.191%,最低检测限为7.2×10~2 copies/μL。山羊IL-2基因的mRNA转录量在0~2 h呈现上升趋势,在2 h达到峰值,2~6 h呈现下降趋势。6~12 h未能检测到IL-2基因的mRNA转录;12~48 h检测到IL-2基因的mRNA转录量呈逐渐上升趋势。研究结果将为山羊IL-2基因的定量分析提供技术平台。
To establish a SYBR GREEN I real-time fluorescence quantitative RT-PCR assay for goat IL-2 gene, a pair of specific primers were designed according to Gen Bank goat IL-2 gene (accession number: KT934548), and used to amplify the target gene. The target gene was cloned into pMD-19-T vector and transformed into E.coli DH5α. Plasmid PCR and sequence analysis showed that the recombinant plasmid was positive. SYBR GREENⅠRTFQ-PCR standard curve and lysis curve were established as templates for standardization. Repeatability and sensitivity tests. And the established method was used to detect the transcription of IL-2 gene at different time points (0, 2, 4, 6, 8, 10, 12, 24 and 48 h after Con A stimulated healthy goats PBMCs) Dynamic changes. The results showed that when the dilution of plasmid standard was between 7.2 × 10 ~ 9 ~ 7.2 × 10 ~ 5 copies / μL, there was a good linear relationship between the Ct value and the concentration, the correlation coefficient was -0.996; the melting curve was specific The coefficient of variation (CV) was 0.306% ~ 1.458% in single group. The coefficient of variation (CV) was 0.514% ~ 1.191%. The lowest detection limit was 7.2 × 10 ~ 2 copies / μL. The transcription level of IL-2 mRNA in goats increased from 0 to 2 h, peaked at 2 h, and decreased from 2 to 6 h. The transcription of IL-2 mRNA was not detected from 6 to 12 h. The mRNA transcription of IL-2 increased gradually from 12 to 48 h. The results will provide a technological platform for the quantitative analysis of goat IL-2 gene.