CAJ | 학술논문

目的构建携带小鼠血管生成素-1（Ang-1）基因的慢病毒载体并进行慢病毒包装。方法利用Gateway技术构建携带DsRed荧光蛋白的Ang-1慢病毒载体，经PCR及基因测序鉴定后，与辅助质粒pLV/helper-SL3、pLV/helper-SL4及pLV/helper-SL5混合采用脂质体法制备DNA-Lipofectamine 2000复合物，并共同转染293FT细胞进行慢病毒包装，产生相应慢病毒颗粒，通过荧光表达情况来测定病毒滴度和感染效率。结果通过PCR、基因测序证实，Ang-1慢病毒表达载体PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2构建成功。经转染293FT细胞包装出具高效感染力的慢病毒，经测定病毒滴度为5×108 TU/ml。结论成功构建Ang-1慢病毒表达载体，并包装出具高效感染力的慢病毒颗粒，为进一步研究Ang-1基因功能及基因治疗提供实验基础。
목적구건휴대소서혈관생성소-1（Ang-1）기인적만병독재체병진행만병독포장。방법이용Gateway기술구건휴대DsRed형광단백적Ang-1만병독재체，경PCR급기인측서감정후，여보조질립pLV/helper-SL3、pLV/helper-SL4급pLV/helper-SL5혼합채용지질체법제비DNA-Lipofectamine 2000복합물，병공동전염293FT세포진행만병독포장，산생상응만병독과립，통과형광표체정황래측정병독적도화감염효솔。결과통과PCR、기인측서증실，Ang-1만병독표체재체PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2구건성공。경전염293FT세포포장출구고효감염력적만병독，경측정병독적도위5×108 TU/ml。결론성공구건Ang-1만병독표체재체，병포장출구고효감염력적만병독과립，위진일보연구Ang-1기인공능급기인치료제공실험기출。
Objective To construct a lentiviral vector carrying angiopoietin-1 (Ang-1) and DsRed gene, and to package a virus particles. Methods The Ang-1 lentiviral vector with DsRed (PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2) was constructed by Gateway technology, and identiifed by PCR and gene sequencing. The lentiviral vector was mixed with helper vector pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5 by Lipofectamine 2000 to prepare DNA-Lipofectamine?2000 complexes. The complexes were then added to transfect 293FT cells and package virus. The virus titers and infection ef-ifciency were determined by lfuorescence expression. Results Ang-1 lentiviral vector PLV.Ex3d.P/puro-CMV>Ang-1>IRES/DsRed-Express2 was constructed successfully as identified by PCR and gene sequencing. Lentivirus with high-efficiency infection was produced by transfection to 293FT cells and the virus titer was 5×108 TU/ml. Conclusions The recombinant len-tiviral vector for Ang-1 was successfully constructed by Gateway technology and the lentivirus with high-efifciency infection packaging can be used for further experiment of Ang-1 gene.