应用实时荧光定量RT-PCR检测猪HEV ORF2基因3个连续片段的真核表达水平  

Detecting eukaryotic expression level of swine hepatitis E virus ORF2 gene three continuous fragments by real-time fluorescent quantification RT-PCR assay

作　　者：苏乾莲[1] 李斌[1] 赵武[1] 秦毅斌[1] 梁家幸[1] 卢冰霞[1] 何颖[1] 段群棚[1] 卢敬专 蒋冬福[1] 周英宁[1] 梁保忠[1] 

机构地区：[1]广西兽医研究所广西畜禽疫苗新技术重点实验室,广西南宁530001

出　　处：《中国兽医学报》2014年第11期1709-1715,共7页Chinese Journal of Veterinary Science

基　　金：广西科技攻关项目(桂科攻10100014-4);广西基本科研业务费专项基金资助项目(桂科专项14-2);广西畜禽疫苗新技术重点实验室系统性研究课题资助项目(12-071-28-A-5)

摘　　要：将构建的猪戊型肝炎病毒广西株3个连续ORF2基因片段重组真核表达质粒pEASY-LB1、pEASY-LB2、pEASY-LB3转染人肾上皮细胞系293T,应用实时荧光定量RT-PCR检测目的基因LB1、LB2、LB3。经ABI7500系统建立的标准曲线相关系数(R2)范围为0.994 209~0.999 130,斜率范围-3.469 403^-3.579 107,扩增效率范围为90.28%~94.19%。经对转染细胞、空白对照细胞的目的基因、内参基因的扩增曲线和融解曲线分析,表明该实时荧光定量PCR扩增体系的特异性良好,通过2-ΔΔCt相对定量法计算,可得出转染细胞293T-a、293T-b、293Tc目的基因LB1、LB2、LB3的基因表达量分别为6 226 830.88±826 430.14、9 449 238.68±926 057.13、35 177 915.63±3 949 173.42。本实验室建立的3个重组真核表达质粒在293T细胞中的转染成功为深入研究猪戊型肝炎基因工程疫苗奠定了基础。The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, pEASY-LB3 were transfected into 293T cells and then three target genes of LB1, LB2, LB3 were detected by realtime fluorescent quantitative RT-PCR. The standard curve was established through ABI7500 Se- quence Detection System. The correlation coefficient（R2） range was between 0. 994 209 and 0. 999 130. The slope value of the standard curves range was between --3. 469 403 and --3. 579 107.7he efficiency of the PCR range was between 90.28% and 94.19%. Analysising the amplificatiol, plot and melting curve of target gene and reference genes, showed that the real-time fluorescent quantita- tive PCR amplification system had good specificity. Using 2-azsct relative quantitative method,tile gene expression quantity of LB1, LB2, LB3 were 6 226 830. 88± 826 430. 14,9 449 238. 68 ±926 057.13,35 177 915.63±3 949 173.42 respectively. Successfully transfected three eukaryotic expression plasmids into 293T cells, which laid the foundation for the further study of swine hepatitis E genetic engineering vaccine.

关 键 词：猪戊型肝炎 真核表达质粒 转染 实时荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]