CAJ | 학술논문

目的：高效包装不同启动子的重组慢病毒（ LV ），建立LV 的定量方法。方法采用分子克隆方法获得pFUGW、pTY-EF1α-EGFP及pTY-CMV-EGFP 3种转移质粒，然后采用脂质体法与另两种包装质粒pSPAX2及pMD2．G共同转染293T细胞，制备含有不同启动子的LV。设计针对3’-LTR的探针和引物，建立LV的荧光定量PCR定量方法。结果获得了不同启动子的转移质粒，制备了3种不同启动子的LV，应用荧光定量PCR方法测定制备的病毒，载量能达到109 copies／ml。结论成功构建了不同启动子的转移质粒，获得了三种LV，建立了针对LV的荧光定量PCR方法，为后续研究不同启动子在多种细胞系中驱动目的蛋白的表达奠定了基础。
목적：고효포장불동계동자적중조만병독（ LV ），건립LV 적정량방법。방법채용분자극륭방법획득pFUGW、pTY-EF1α-EGFP급pTY-CMV-EGFP 3충전이질립，연후채용지질체법여령량충포장질립pSPAX2급pMD2．G공동전염293T세포，제비함유불동계동자적LV。설계침대3’-LTR적탐침화인물，건립LV적형광정량PCR정량방법。결과획득료불동계동자적전이질립，제비료3충불동계동자적LV，응용형광정량PCR방법측정제비적병독，재량능체도109 copies／ml。결론성공구건료불동계동자적전이질립，획득료삼충LV，건립료침대LV적형광정량PCR방법，위후속연구불동계동자재다충세포계중구동목적단백적표체전정료기출。
Objective To package recombinant lentivirus ( LV) efficiently and establish quantitative method for LV .Methods Firstly, gene cloning method was used to construct three transfer plasmids pFUGW , pTY-EF1α-EGFP and pTY-CMV-EGFP and then 293T cells were co-transfected with packaging plasmids pSPAX 2 and pMD2.G by liposome, lentivirus with different promoters were obtained.The probe and primers aimed at 3’-LTR were designed and the fluorescent quantitative method for LV was set up .Re-sults Transfer plasmids with different promoters were obtained ,LV with three different promoters was manufactured ,the QPCR method worked well and lentivirus load could achieve 109 copies/ml by it.Conclusion Transfer plasmids with different promoters have been successfully constructed , three kinds of lentivirus have been acquired and the QPCR method for LV has been established , which has laid the foundation for the follow-up study of different promoters driven protein expression among various cells .