CAJ | 학술논문

目的构建pLVX‐IRES‐TDtomat‐GSK‐3β慢病毒表达载体，感染小鼠骨髓DC2．4细胞，建立过表达GSK‐3β的小鼠DC2．4细胞系。方法利用RT‐PCR方法扩增小鼠GSK‐3β基因的编码区，并将其重组于表达载体pLVX‐IRES‐TDtomat中，经酶切、测序鉴定后包装成慢病毒，感染小鼠DC2．4细胞。实验分3组，分别为DC2．4细胞对照组，空病毒感染组，GSK‐3β慢病毒感染组，用实时荧光定量PCR方法和Western Blotting方法检测各组GSK‐3β的表达水平。结果经酶切和测序鉴定结果证实pLVX‐IRES‐ TDtomat‐GSK‐3β构建成功，并在包装细胞中高水平表达，慢病毒滴度高达1．26×108 TU／mL。GSK‐3β慢病毒感染组的GSK‐3β在mRNA表达水平和蛋白表达水平上均高于DC2．4细胞对照组及空病毒感染组。结论成功构建了表达GSK3β基因的重组慢病毒载体，并可在DC2．4细胞中稳定表达，为后续DC2．4的免疫功能研究奠定了基础。
목적구건pLVX‐IRES‐TDtomat‐GSK‐3β만병독표체재체，감염소서골수DC2．4세포，건립과표체GSK‐3β적소서DC2．4세포계。방법이용RT‐PCR방법확증소서GSK‐3β기인적편마구，병장기중조우표체재체pLVX‐IRES‐TDtomat중，경매절、측서감정후포장성만병독，감염소서DC2．4세포。실험분3조，분별위DC2．4세포대조조，공병독감염조，GSK‐3β만병독감염조，용실시형광정량PCR방법화Western Blotting방법검측각조GSK‐3β적표체수평。결과경매절화측서감정결과증실pLVX‐IRES‐ TDtomat‐GSK‐3β구건성공，병재포장세포중고수평표체，만병독적도고체1．26×108 TU／mL。GSK‐3β만병독감염조적GSK‐3β재mRNA표체수평화단백표체수평상균고우DC2．4세포대조조급공병독감염조。결론성공구건료표체GSK3β기인적중조만병독재체，병가재DC2．4세포중은정표체，위후속DC2．4적면역공능연구전정료기출。
[Objective]To construct pLVX‐IRES‐TDtomat‐GSK‐3βlentiviral expression vectors and infect murine bone marrow DC2 .4 cell line for establishing DC2 .4 cell line with an over‐expression of murine GSK‐3β.[Methods]Coding sequence in murine GSK‐3β cDNA was amplified by reverse transcription‐polymerase chain reaction (RT‐PCR) assay and recombined into pLVX‐IRES‐TDtomat plasmid .After confirming with re‐striction endonucleases and sequencing ,the recombinant plasmid was transfected into 293T cells with Lipo‐fectamine 2000 and then packaged into lentivirus particles .DC2 .4 cell line was infected by lentivirus particles . There were 3 groups of DC2 .4 cell line control ,non‐virus control and GSK‐3βlentivirus .The expression lev‐els of GSK‐3βamong three groups were detected by real‐time PCR and Western blot .[Results]Restriction en‐donuclease assay and sequence analysis verified the successful construction of recombinant vector pLVX‐IRES‐TDtomat‐GSK‐3β.And it was expressed highly in packaging cell 293T and the titer of recombinant lentivirus particles was 1 .26 × 108 TU/mL .GSK‐3βexpression level increased more in GSK‐3βlentivirus group than that in DC2 .4 cell line control and non‐virus control at the levels of mRNA and protein .[Conclusion]GSK‐3βlenti‐viral recombinant vector is successfully constructed and expressed stably in DC2 .4 cell line .The study may provide rationales for further elucidating the immunological functions of DC2 .4 .