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当前位置: 首页 > 文章 > 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 中国兽医学报 2014,34 (11) 1709-1715
Position: Home > Articles > Detecting eukaryotic expression level of swine hepatitis E virus ORF2 gene three continuous fragments by real-time fluorescent quantification RT-PCR assay Chinese Journal of Veterinary Science 2014,34 (11) 1709-1715

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

作  者：
苏乾莲;李斌;赵武;秦毅斌;梁家幸;卢冰霞;何颖;段群棚;卢敬专;蒋冬福;周英宁;梁保忠
单  位：
广西兽医研究所广西畜禽疫苗新技术重点实验室
关键词：
猪戊型肝炎;真核表达质粒;转染;实时荧光定量PCR
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。
译  名：
Detecting eukaryotic expression level of swine hepatitis E virus ORF2 gene three continuous fragments by real-time fluorescent quantification RT-PCR assay
作  者：
SU Qian-lian;LI Bin;ZHAO Wu;QIN Yi-bin;LIANG Jia-xing;LU Bing-xia;HE Ying;DUAN Qun-peng;LU Jing-zhuan;JIANG Dong-fu;ZHOU Ying-ning;LIANG Bao-zhong;Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Animal Vaccines and New Technology;
关键词：
swine hepatitis E;;eukaryotic expression;;transfect;;real-time fluorescent quantitative RT-PCR
摘  要：
The eukaryotic expression plasmids of pEASY-LB1,pEASY-LB2,pEASY-LB3 were transfected into 293 Tcells and then three target genes of LB1,LB2,LB3 were detected by realtime fluorescent quantitative RT-PCR.The standard curve was established through ABI7500 Sequence 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 403and-3.579 107.The efficiency of the PCR range was between 90.28%and 94.19%.Analysising the amplification plot and melting curve of target gene and reference genes,showed that the real-time fluorescent quantitative PCR amplification system had good specificity.Using 2-ΔΔCt relative quantitative method,the 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 293 Tcells,which laid the foundation for the further study of swine hepatitis E genetic engineering vaccine.

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