猪源MHC Ⅱ类分子反式激活因子CIITA SYBR Green Ⅰ荧光定量PCR检测方法的建立  

Development of A SYBR Green Ⅰ Real-time PCR for Detection of Porcine MHC Class Ⅱ Gene Transactivator CIITA

作　　者：朱豪杰 高飞[1] 李丽薇[1] 虞凌雪[1] 张玉娇 张宽 童光志[1] 姜一峰[1] 李国新[1,2] ZHU Haojie;GAO Fei;LI Liwei;YU Lingxue;ZHANG Yujiao;ZHANG Kuan;TONG Guangzhi;JIANG Yifeng;LI Guoxin(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou 225009,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2022年第2期107-114,共8页Chinese Journal of Animal Infectious Diseases

摘　　要：Class Ⅱ Transactivator(CIITA)作为重要的MHCⅡ类分子反式激活因子在抗原递呈细胞的成熟和功能实现过程中发挥关键作用,为建立一种快速检测猪源CIITA基因的方法,本研究根据GenBank中猪源CIITA基因序列设计引物,从猪肺泡巨噬细胞的总cDNA中扩增CIITA基因,并克隆到pCold-TF载体中获得重组质粒pCold-TF-CIITA。利用重组质粒pCold-TF-CIITA作为标准品建立SYBR Green Ⅰ荧光定量PCR检测方法。试验结果显示:该方法在1.68×10^(1)~1.68×10^(7) copies/μL范围内呈现出良好的线性关系,检测灵敏度可达16.8 copies/μL,组内和组间变异系数均小于1%。利用该方法对不同细胞中的CIITA基因进行检测,结果显示该方法仅能够检测到猪源细胞中的CIITA基因。本试验结果表明,利用pCold-TF-CIITA作为标准品建立的CIITA基因SYBR Green Ⅰ荧光定量PCR检测方法具有良好的特异性、敏感性和重复性,可用于CIITA基因的定量检测。As an important MHC class Ⅱ Gene transactivator,Class Ⅱ Transactivator(CIITA)plays a key role in maturation and function realization of antigen-presenting cells.In order to develop a rapid detection method for porcine CIITA,the CIITA gene was amplified from the total cDNA of alveolar macrophages and then cloned into the pCold-TF vector,the recombinant plasmid pCold-TF-CIITA was used as a standard sample and a SYBR Green Ⅰ quantitative PCR assay was developed.The results showed that a linear relationship ranged from 1.68×10^(1)~1.68×10^(7) copies/μL of stand samples and the sensitivity of the detected standard was 16.8 copies/μL.The intra-group and inter-group coefficients of variation were both less than 1% and had no cross-reactivity with the CIITA genes from other species.In summary,the SYBR Green Ⅰ real-time quantitative PCR assay based on CIITA standards provided an efficient diagnostic method for the quantitative detection of porcine CIITA gene.

关 键 词：猪源CIITA 荧光定量PCR SYBR GreenⅠ 

分 类 号：Q78[生物学—分子生物学] S852.4[农业科学—基础兽医学]