小鼠GSK-3β慢病毒表达载体的构建及其在骨髓树突状细胞DC2.4中的表达  

Construction of Murine GSK3β Lentiviral Expression Vector and its Expression in DC2.4 Cell Line

作　　者：包杰[1] 熊石龙[2] 

机构地区：[1]广东医学院附属医院检验科,广东湛江524001 [2]广州医科大学第四附属医院检验科,广东广州511447

出　　处：《医学临床研究》2015年第1期13-16,共4页Journal of Clinical Research

基　　金：国家自然科学基金

摘　　要：【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，GSK313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-313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1．26×108Tu／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GSK313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。[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-3β lentivirus. The expression lev- els of GSK-3β among three groups were detected by real-time PCR and Western blot. [Resultsl 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-3β expression level increased more in GSK-3β lentivirus group than that in DC2.4 cell line control and non-virus control at the levels of mRNA and protein. [Conclusion]GSK-3β lenti- 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.

关 键 词：蛋白激酶类 骨髓细胞 树突细胞/免疫学 慢病毒属/遗传学 遗传载体 

分 类 号：R394.2[医药卫生—医学遗传学]