CAJ | 학술논문

为探讨PRRSV感染后机体的体液免疫应答、从分子水平深入研究PRRSV的免疫机制,本研究针对Th2型细胞因子IL-4、IL-6、IL-10以及看家基因β-actin的基因序列分别设计一对特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测IL-4、IL-6、IL-10及β-actin的SYBR Green Ⅰ real-time PCR方法.该方法线性关系好,各种细胞因子及β-actin标准曲线的相关系数均达到0.997以上;敏感性高,初始模板的检出下限均达到1×10~1 copies/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内与组间的变异系数均小于3%.应用所建立的方法对猪繁殖与呼吸综合征病毒(PRRSV)感染仔猪外周血单个核细胞(PBMC)中IL-4、IL-6和IL-10 mRNA的表达水平进行了检测.结果表明,本研究建立的real-time PCR检测方法灵敏度高、特异性强、重复性好,可以用于猪Th2型细胞因子的检测及定量分析.
위탐토PRRSV감염후궤체적체액면역응답、종분자수평심입연구PRRSV적면역궤제,본연구침대Th2형세포인자IL-4、IL-6、IL-10이급간가기인β-actin적기인서렬분별설계일대특이성인물,구건함유각자인물확증서렬적중조질립작위양성표준품,건립료검측IL-4、IL-6、IL-10급β-actin적SYBR Green Ⅰ real-time PCR방법.해방법선성관계호,각충세포인자급β-actin표준곡선적상관계수균체도0.997이상;민감성고,초시모판적검출하한균체도1×10~1 copies/μL;특이성강,확증산물형성단일적특이성용해봉;중복성호,조내여조간적변이계수균소우3%.응용소건립적방법대저번식여호흡종합정병독(PRRSV)감염자저외주혈단개핵세포(PBMC)중IL-4、IL-6화IL-10 mRNA적표체수평진행료검측.결과표명,본연구건립적real-time PCR검측방법령민도고、특이성강、중복성호,가이용우저Th2형세포인자적검측급정량분석.
Real-time PCR assays based on SYBR Green Ⅰ for detection of IL-4, IL-6, IL-10 and β-actin were established us-ing primers derived from porcine Th2-type cytokines (IL-4, IL-6 and IL-10) gene. The assays were highly sensitive and had a de-tection limit of 1×10~1 copies/μL of initial templates. These assays were highly specific and there was single specific melting peak for every cytokine. It was highly reproducible and had a coefficient of variation less than 3 percent for both intra-and inter-assay. The established assays were successfully used to detect IL-4, IL-6 and IL-10 mRNA expression levels in peripheral blood mononu-clear cells (PBMCs) in piglets experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The high sensitivity, specificity and reproducibility of the assays indicated that the SYBR Green Ⅰ real-time PCR could be used as an effective tool for detection and quantification of Th2-type cytokines.